46 results on '"Barnidge DR"'
Search Results
2. A design for low-flow sheathless electrospray emitters
- Author
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Barnidge, DR, Nilsson, S, Markides, KE, Barnidge, DR, Nilsson, S, and Markides, KE
- Abstract
An extremely simple design has been developed for producing durable sheathless electrospray emitters that give highly stable electrospray for unlimited lifetimes. The emitters can be fashioned from any style fused-silica capillary and are ideally suited, Addresses: Markides KE, Univ Uppsala, Dept Analyt Chem, Box 531, S-75121 Uppsala, Sweden. Univ Uppsala, Dept Analyt Chem, S-75121 Uppsala, Sweden.
- Published
- 1999
3. Analysis of monoclonal immunoglobulins from bone marrow plasma cells using immunopurification and LC-MS.
- Author
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Barnidge DR, Dispenzieri A, Jevremovic D, and Murray DL
- Abstract
Introduction: Clonal plasma cells secrete immunoglobulins, each with the exact same amino acid sequence, that are referred to as monoclonal immunoglobulins. The monoclonal heavy chain and light chain secreted from clonal plasma cells have the same molecular mass prior to the addition of post-translational modifications (PTMs) since their amino acid sequences are the same., Objective: To examine the molecular masses of monoclonal light chains and heavy chains isolated directly from the cytoplasm of bone marrow (BM) plasma cells and compare them to the serum derived monoclonal heavy and light chains., Methods: Using immunopurification and LC-MS we compared the molecular masses of immunoglobulins immunopurified from a patient's serum to those immunopurified from the cytoplasm of their BM plasma cells., Results: Our findings demonstrate that the light chain molecular masses were identical whether they were obtained from serum or plasma cell cytoplasm. However, the heavy chain molecular masses did not match in bone marrow and serum due to differences in glycosylation, a common post-translational modification (PTM) found on the heavy chain., Conclusion: The data presented here show that by using LC-MS to analyze monoclonal immunoglobulins (also referred to as miRAMM) additional phenotype information is obtained at the cellular level which is complementary to other more common techniques such as flow cytometry and histopathology., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 THE AUTHORS.)
- Published
- 2023
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4. Classification of Plasma Cell Disorders by 21 Tesla Fourier Transform Ion Cyclotron Resonance Top-Down and Middle-Down MS/MS Analysis of Monoclonal Immunoglobulin Light Chains in Human Serum.
- Author
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He L, Anderson LC, Barnidge DR, Murray DL, Dasari S, Dispenzieri A, Hendrickson CL, and Marshall AG
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- Amino Acid Sequence, Amyloidosis diagnosis, Antibodies, Monoclonal analysis, Antibodies, Monoclonal metabolism, Chromatography, High Pressure Liquid, Fourier Analysis, Humans, Immunoglobulin Light Chains chemistry, Immunoglobulins isolation & purification, Immunoglobulins metabolism, Multiple Myeloma diagnosis, Paraproteinemias classification, Paraproteinemias diagnosis, Amyloidosis classification, Immunoglobulin Light Chains blood, Multiple Myeloma classification, Tandem Mass Spectrometry methods
- Abstract
The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is ∼51%, indicating the urgent need for better diagnosis methods and treatment plans. Here, we describe highly specific and sensitive top-down and middle-down MS/MS methods owning the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages with 21 telsa FT-ICR MS/MS. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by database-aided de novo MS/MS and 100% matched the gene sequencing result, except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. The monoclonal immunoglobulin heavy chains were also classified into specific germline sequences based on the present method. This work represents the first application of top/middle-down MS/MS sequencing of endogenous human monoclonal immunoglobulins with polyclonal immunoglobulins background.
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- 2019
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5. Pharmacokinetics of rituximab and clinical outcomes in patients with anti-neutrophil cytoplasmic antibody associated vasculitis.
- Author
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Cornec D, Kabat BF, Mills JR, Cheu M, Hummel AM, Schroeder DR, Cascino MD, Brunetta P, Murray DL, Snyder MR, Fervenza F, Hoffman GS, Kallenberg CGM, Langford CA, Merkel PA, Monach PA, Seo P, Spiera RF, St Clair EW, Stone JH, Barnidge DR, and Specks U
- Subjects
- Adult, Aged, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis blood, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis immunology, B-Lymphocytes immunology, B-Lymphocytes pathology, Dose-Response Relationship, Drug, Double-Blind Method, Female, Humans, Immunologic Factors administration & dosage, Immunologic Factors pharmacokinetics, Infusions, Intravenous, Lymphocyte Count, Male, Middle Aged, Remission Induction, Rituximab administration & dosage, Treatment Outcome, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis drug therapy, Rituximab pharmacokinetics
- Abstract
Objectives: To study the determinants of the pharmacokinetics (PK) of rituximab (RTX) in patients with ANCA-associated vasculitis (AAV) and its association with clinical outcomes., Methods: This study included data from 89 patients from the RTX in AAV trial who received the full dose of RTX (four weekly infusions of 375 mg/m2). RTX was quantified at weeks 2, 4, 8, 16 and 24, and summarized by computing the trapezoidal area under the curve. We explored potential determinants of the PK-RTX, and analysed its association with clinical outcomes: achievement of remission at 6 months, duration of B-cell depletion and time to relapse in patients who achieved complete remission., Results: RTX serum levels were significantly lower in males and in newly diagnosed patients, and negatively correlated with body surface area, baseline B-cell count and degree of disease activity. In multivariate analyses, the main determinants of PK-RTX were sex and new diagnosis. Patients reaching complete remission at month 6 had similar RTX levels compared with patients who did not reach complete remission. Patients with higher RTX levels generally experienced longer B-cell depletion than patients with lower levels, but RTX levels at the different time points and area under the curve were not associated with time to relapse., Conclusion: Despite the body-surface-area-based dosing protocol, PK-RTX is highly variable among patients with AAV, its main determinants being sex and newly diagnosed disease. We did not observe any relevant association between PK-RTX and clinical outcomes. The monitoring of serum RTX levels does not seem clinically useful in AAV.
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- 2018
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6. High sensitivity blood-based M-protein detection in sCR patients with multiple myeloma.
- Author
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Mills JR, Barnidge DR, Dispenzieri A, and Murray DL
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- Adult, Aged, Autografts, Female, Humans, Immunoglobulin G blood, Male, Mass Spectrometry, Middle Aged, Remission Induction, Retrospective Studies, Glycoproteins blood, Multiple Myeloma blood, Multiple Myeloma therapy, Stem Cell Transplantation
- Abstract
We assessed the ability of a mass spectrometry-based technique, called monoclonal immunoglobulin rapid accurate mass measurement (miRAMM), to extend the analytical range of M-protein detection in serum samples obtained from myeloma patients in stringent complete response (sCR) post-autologous stem cell transplant (ASCT). To aid the M-protein detection post ASCT, the accurate molecular mass of the M-protein light chain at diagnosis was determined in all patients (N=30) and used to positively identify clones in the sCR serum. Day 100 post-ASCT, sCR samples had miRAMM identifiable M-proteins in 81% of patients. Patients who had achieved only a partial remission (PR) pre-ASCT and those with IgG isotypes serum samples had the highest rate of M-protein detection by miRAMM. miRAMM positivity at single time points (day 100, 6 months or 12 months) did not correlate with progression-free survival (PFS). In contrast, sCR patients who did not decrease their miRAMM M-protein intensities in serial measurements had shorter PFS than those whose miRAMM intensities decreased (median 17.9 months vs 51.6 months; P<0.0017). miRAMM M-protein is a more sensitive blood-based test than traditional M-protein tests and could cost effectively aid in serially monitoring complete remission for continue response or biochemical relapse.
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- 2017
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7. The utility of MASS-FIX to detect and monitor monoclonal proteins in the clinic.
- Author
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Milani P, Murray DL, Barnidge DR, Kohlhagen MC, Mills JR, Merlini G, Dasari S, and Dispenzieri A
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- Aged, Biomarkers, Diagnosis, Differential, Female, Humans, Male, Middle Aged, Prospective Studies, Sensitivity and Specificity, Multiple Myeloma blood, Multiple Myeloma diagnosis, Myeloma Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
The detection and quantification of monoclonal-proteins (M-proteins) are necessary for the diagnosis and evaluation of response in plasma cell dyscrasias. Immunoglobulin enrichment-coupled with matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MASS-FIX) is a simple and inexpensive method to identify M-proteins, but its clinical generalizability has not yet been elucidated. We compared MASS-FIX to protein electrophoresis (PEL), serum/urine immunofixation-electrophoresis (IFE), and quantitative serum free-light chain (FLC) for the identification of M-proteins in different clinical diagnoses. Paired serum and urine samples from 257 patients were tested. There were six patients for whom s-IFE and FLC ratio were positive and serum MASS-FIX was negative, but when serum and urine MASS-FIX results were combined, only one patient with light chain-MGUS was missed. Serum/urine-MASS-FIX detected M-proteins in 18 patients with negative serum/urine-PEL/IFE and serum-FLC, 10 of whom had multiple myeloma or AL amyloidosis, who were mistakenly thought to have complete hematologic response by serum/urine-PEL/IFE and serum-FLC. Nearly half of the AL amyloidosis patients had atypical spectra, which may prove to be a clue to the diagnosis and pathogenesis of the disease. In conclusion, MASS-FIX has a comparable sensitivity with PEL/IFE/FLC methods and can help inform the clinical diagnosis., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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8. Assessment of renal response with urinary exosomes in patients with AL amyloidosis: A proof of concept.
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Ramirez-Alvarado M, Barnidge DR, Murray DL, Dispenzieri A, Marin-Argany M, Dick CJ, Cooper SA, Nasr SH, Ward CJ, Dasari S, Jiménez-Zepeda VH, and Leung N
- Subjects
- Adult, Aged, Amino Acid Sequence, Amyloidosis genetics, Female, Humans, Immunoglobulin Light Chains chemistry, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains urine, Immunoglobulin Light-chain Amyloidosis, Male, Mass Spectrometry, Middle Aged, Molecular Weight, Protein Aggregates, Protein Aggregation, Pathological metabolism, Protein Aggregation, Pathological urine, Sequence Analysis, DNA, Amyloidosis complications, Amyloidosis metabolism, Exosomes metabolism, Immunoglobulin Light Chains metabolism, Proteinuria diagnosis, Proteinuria etiology
- Abstract
Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future., (© 2017 Wiley Periodicals, Inc.)
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- 2017
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9. Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory.
- Author
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Ladwig PM, Barnidge DR, and Willrich MAV
- Subjects
- Humans, Antibodies, Monoclonal analysis, Mass Spectrometry methods
- Abstract
Therapeutic monoclonal antibodies (MAbs) are an important class of drugs used to treat diseases ranging from autoimmune disorders to B cell lymphomas to other rare conditions thought to be untreatable in the past. Many advances have been made in the characterization of immunoglobulins as a result of pharmaceutical companies investing in technologies that allow them to better understand MAbs during the development phase. Mass spectrometry is one of the new advancements utilized extensively by pharma to analyze MAbs and is now beginning to be applied in the clinical laboratory setting. The rise in the use of therapeutic MAbs has opened up new challenges for the development of assays for monitoring this class of drugs. MAbs are larger and more complex than typical small-molecule therapeutic drugs routinely analyzed by mass spectrometry. In addition, they must be quantified in samples that contain endogenous immunoglobulins with nearly identical structures. In contrast to an enzyme-linked immunosorbent assay (ELISA) for quantifying MAbs, mass spectrometry-based assays do not rely on MAb-specific reagents such as recombinant antigens and/or anti-idiotypic antibodies, and time for development is usually shorter. Furthermore, using molecular mass as a measurement tool provides increased specificity since it is a first-order principle unique to each MAb. This enables rapid quantification of MAbs and multiplexing. This review describes how mass spectrometry can become an important tool for clinical chemists and especially immunologists, who are starting to develop assays for MAbs in the clinical laboratory and are considering mass spectrometry as a versatile platform for the task., (Copyright © 2017 Ladwig et al.)
- Published
- 2017
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10. Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry.
- Author
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Ladwig PM, Barnidge DR, and Willrich MA
- Subjects
- Antibodies, Monoclonal, Humanized analysis, Antibodies, Monoclonal, Humanized isolation & purification, Humans, Immunoglobulin G isolation & purification, Limit of Detection, Reproducibility of Results, Antibodies, Monoclonal, Humanized blood, Chromatography, Affinity methods, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure. Graphical Abstract ᅟ.
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- 2017
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11. Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS.
- Author
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He L, Anderson LC, Barnidge DR, Murray DL, Hendrickson CL, and Marshall AG
- Subjects
- Adalimumab analysis, Amino Acid Sequence, Antibodies, Monoclonal analysis, Fourier Analysis, Humans, Protein Processing, Post-Translational, Software, Spectrometry, Mass, Electrospray Ionization methods, Adalimumab blood, Antibodies, Monoclonal blood, Paraproteinemias blood, Tandem Mass Spectrometry methods
- Abstract
With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Graphical Abstract ᅟ.
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- 2017
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12. Erratum to: Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS.
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He L, Anderson LC, Barnidge DR, Murray DL, Hendrickson CL, and Marshall AG
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- 2017
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13. Screening Method for M-Proteins in Serum Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.
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Kohlhagen MC, Barnidge DR, Mills JR, Stoner J, Gurtner KM, Liptac AM, Lofgren DI, Vanderboom PM, Dispenzieri A, Katzmann JA, Willrich MA, Snyder MR, and Murray DL
- Subjects
- Antibodies, Monoclonal immunology, Humans, Immunoglobulin Light Chains immunology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Antibodies, Monoclonal blood, Immunoglobulin Light Chains blood, Nanoparticles chemistry
- Abstract
Background: Current recommendations for screening for monoclonal gammopathies include serum protein electrophoresis (PEL), imunofixation electrophoresis (IFE), and free light chain (FLC) ratios to identify or rule out an M-protein. The aim of this study was to examine the feasibility of an assay based on immunoenrichment and MALDI-TOF-MS (MASS-SCREEN) to qualitatively screen for M-proteins., Methods: Serum from 556 patients previously screened for M-proteins by PEL and IFE were immunopurified using a κ/λ-specific nanobody bead mixture. Following purification, light chains (LC) were released from their heavy chains by reduction. MALDI-TOF analysis was performed and the mass-to-charge LC distributions were visually examined for the presence of an M-protein by both unblinded and blinded analysts., Results: In unblinded analysis, MASS-SCREEN detected 100% of the PEL-positive samples with an analytical sensitivity and specificity of 96% and 81% using IFE positivity as the standard. In a blinded analysis using 6 different laboratory personnel, consensus was reached in 92% of the samples. Overall analytical sensitivity and specificity were reduced to 92% and 80%, respectively. FLC ratios were found to be abnormal in 28% of MASS-SCREEN-negative samples, suggesting FLC measurements need to be considered in screening., Conclusions: MASS-SCREEN could replace PEL in a panel that would include FLC measurements. Further studies and method development should be performed to validate the clinical sensitivity and specificity and to determine if this panel will suffice as a general screen for monoclonal proteins., (© 2016 American Association for Clinical Chemistry.)
- Published
- 2016
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14. Comprehensive Assessment of M-Proteins Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.
- Author
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Mills JR, Kohlhagen MC, Dasari S, Vanderboom PM, Kyle RA, Katzmann JA, Willrich MA, Barnidge DR, Dispenzieri A, and Murray DL
- Subjects
- Antibodies, Monoclonal immunology, Humans, Immunoglobulin Light Chains immunology, Nanoparticles chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Antibodies, Monoclonal blood, Immunoglobulin Light Chains blood, Nanoparticles analysis
- Abstract
Background: Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests., Methods: We developed a MALDI mass spectrometry-based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays., Results: MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to <1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were <20% across all samples having M-protein concentrations >0.045 g/dL, with the ability to detect M-proteins <0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite(™) assay., Conclusions: MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored., (© 2016 American Association for Clinical Chemistry.)
- Published
- 2016
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15. Using Mass Spectrometry to Quantify Rituximab and Perform Individualized Immunoglobulin Phenotyping in ANCA-Associated Vasculitis.
- Author
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Mills JR, Cornec D, Dasari S, Ladwig PM, Hummel AM, Cheu M, Murray DL, Willrich MA, Snyder MR, Hoffman GS, Kallenberg CG, Langford CA, Merkel PA, Monach PA, Seo P, Spiera RF, St Clair EW, Stone JH, Specks U, and Barnidge DR
- Subjects
- Algorithms, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis drug therapy, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis metabolism, Antibodies immunology, Antibodies, Monoclonal, Humanized blood, Antibodies, Monoclonal, Humanized immunology, Antibodies, Monoclonal, Humanized therapeutic use, Chromatography, High Pressure Liquid, Humans, Limit of Detection, Phenotype, Rituximab immunology, Rituximab therapeutic use, Spectrometry, Mass, Electrospray Ionization, Enzyme-Linked Immunosorbent Assay, Rituximab blood
- Abstract
Therapeutic monoclonal immunoglobulins (mAbs) are used to treat patients with a wide range of disorders including autoimmune diseases. As pharmaceutical companies bring more fully humanized therapeutic mAb drugs to the healthcare market analytical platforms that perform therapeutic drug monitoring (TDM) without relying on mAb specific reagents will be needed. In this study we demonstrate that liquid-chromatography-mass spectrometry (LC-MS) can be used to perform TDM of mAbs in the same manner as smaller nonbiologic drugs. The assay uses commercially available reagents combined with heavy and light chain disulfide bond reduction followed by light chain analysis by microflow-LC-electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). Quantification is performed using the peak areas from multiply charged mAb light chain ions using an in-house developed software package developed for TDM of mAbs. The data presented here demonstrate the ability of an LC-MS assay to quantify a therapeutic mAb in a large cohort of patients in a clinical trial. The ability to quantify any mAb in serum via the reduced light chain without the need for reagents specific for each mAb demonstrates the unique capabilities of LC-MS. This fact, coupled with the ability to phenotype a patient's polyclonal repertoire in the same analysis further shows the potential of this approach to mAb analysis.
- Published
- 2016
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16. Monoclonal antibody therapeutics as potential interferences on protein electrophoresis and immunofixation.
- Author
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Willrich MA, Ladwig PM, Andreguetto BD, Barnidge DR, Murray DL, Katzmann JA, and Snyder MR
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- Antibodies, Monoclonal therapeutic use, Blood Protein Electrophoresis, Diagnostic Errors prevention & control, Humans, Immunoelectrophoresis, Immunoglobulin G blood, Immunoglobulin kappa-Chains blood, Immunoglobulin lambda-Chains blood, Inflammation drug therapy, Antibodies, Monoclonal blood, Paraproteinemias diagnosis
- Abstract
Background: The use of therapeutic recombinant monoclonal antibodies (mAbs) has triggered concerns of mis-diagnosis of a plasma cell dyscrasia in treated patients. The purpose of this study is to determine if infliximab (INF), adalimumab (ADA), eculizumab (ECU), vedolizumab (VEDO), and rituximab (RITU) are detected as monoclonal proteins by serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE)., Methods: Pooled normal sera were spiked with various concentrations (ranging from trough to peak) of INF, ADA, ECU, VEDO and RITU. The peak concentration for VEDO and RITU was also added to samples with known monoclonal gammopathies. All samples were analyzed by SPEP (Helena Laboratories) and IFE (Sebia); sera containing peak concentrations of mAbs were reflexed to electrospray-time-of-flight mass spectrometry (AbSciex Triple TOF 5600) for the intact light chain monoclonal immunoglobulin rapid accurate mass measurement (miRAMM)., Results: For all mAbs tested, no quantifiable M-spikes were observed by SPEP at any concentration analyzed. Small γ fraction abnormalities were noted on SPEP for VEDO at 300 μg/mL and RITU at 400 μg/mL, with identification of small IgG κ proteins on IFE. Using miRAMM for peak samples, therapeutic mAbs light chain accurate masses were identified above the polyclonal background and were distinct from endogenous monoclonal gammopathies., Conclusions: MAbs should not be easily confounded with plasma cell dyscrasias in patients undergoing therapy except when a SPEP and IFE are performed within a couple of days from infusion (peak). In ambiguous cases the use of the miRAMM technology could precisely identify the therapeutic mAb distinct from any endogenous monoclonal protein.
- Published
- 2016
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17. Laboratory testing in monoclonal gammopathy of renal significance (MGRS).
- Author
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Leung N, Barnidge DR, and Hutchison CA
- Subjects
- Blood Protein Electrophoresis, Humans, Immunoassay, Immunoelectrophoresis, Immunoglobulin Light Chains blood, Immunoglobulin Light Chains urine, Kidney Diseases blood, Kidney Diseases etiology, Kidney Diseases urine, Limit of Detection, Myeloma Proteins analysis, Paraproteinemias blood, Paraproteinemias complications, Paraproteinemias urine, Kidney Diseases diagnosis, Paraproteinemias diagnosis, Paraproteins urine
- Abstract
Recently, monoclonal gammopathy of renal significance (MGRS) reclassified all monoclonal (M) gammopathies that are associated with the development of a kidney disease but do not meet the definition of symptomatic multiple myeloma (MM) or malignant lymphoma. The purpose was to distinguish the M gammopathy as the nephrotoxic agent independent from the clonal mass. The diagnosis of MGRS obviously depends on the detection of the M-protein. More importantly, the success of treatment is correlated with the reduction of the M-protein. Therefore, familiarity with the M-protein tests is a must. Protein electrophoresis performed in serum or urine is inexpensive and rapid due to automation. However, poor sensitivity especially with the urine is an issue particularly with the low-level M gammopathy often encountered with MGRS. Immunofixation adds to the sensitivity and specificity but also the cost. Serum free light chain (sFLC) assays have significantly increased the sensitivity of M-protein detection and is relatively inexpensive. It is important to recognize that there is more than one assay on the market and their results are not interchangeable. In addition, in certain diseases, immunofixation is more sensitive than sFLC. Finally, novel techniques with promising results are adding to the ability to identify M-proteins. Using the time of flight method, the use of mass spectrometry of serum samples has been shown to dramatically increase the sensitivity of M-protein detection. In another technique, oligomeric LCs are identified on urinary exosomes amplifying the specificity for the nephrotoxic M-protein.
- Published
- 2016
- Full Text
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18. Monitoring free light chains in serum using mass spectrometry.
- Author
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Barnidge DR, Dispenzieri A, Merlini G, Katzmann JA, and Murray DL
- Subjects
- Adalimumab blood, Amyloidosis blood, Amyloidosis immunology, Humans, Immunoglobulin Heavy Chains blood, Immunoglobulin kappa-Chains urine, Immunoglobulin lambda-Chains urine, Spectrometry, Mass, Electrospray Ionization, Immunoglobulin kappa-Chains blood, Immunoglobulin lambda-Chains blood
- Abstract
Background: Serum immunoglobulin free light chains (FLC) are secreted into circulation by plasma cells as a by-product of immunoglobulin production. In a healthy individual the population of FLC is polyclonal as no single cell is secreting more FLC than the total immunoglobulin secreting cell population. In a person with a plasma cell dyscrasia, such as multiple myeloma (MM) or light chain amyloidosis (AL), a clonal population of plasma cells secretes a monoclonal light chain at a concentration above the normal polyclonal background., Methods: We recently showed that monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) can be used to identify and quantify a monoclonal light chain (LC) in serum and urine above the polyclonal background. This was accomplished by reducing immunoglobulin disulfide bonds releasing the LC to be analyzed by microLC-ESI-Q-TOF mass spectrometry. Here we demonstrate that the methodology can also be applied to the detection and quantification of FLC by analyzing a non-reduced sample., Results: Proof of concept experiments were performed using purified FLC spiked into normal serum to assess linearity and precision. In addition, a cohort of 27 patients with AL was analyzed and miRAMM was able to detect a monoclonal FLC in 23 of the 27 patients that had abnormal FLC values by immunonephelometry., Conclusions: The high resolution and high mass measurement accuracy provided by the mass spectrometry based methodology eliminates the need for κ/λ ratios as the method can quantitatively monitor the abundance of the κ and λ polyclonal background at the same time it measures the monoclonal FLC.
- Published
- 2016
- Full Text
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19. Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.
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Hoofnagle AN, Whiteaker JR, Carr SA, Kuhn E, Liu T, Massoni SA, Thomas SN, Townsend RR, Zimmerman LJ, Boja E, Chen J, Crimmins DL, Davies SR, Gao Y, Hiltke TR, Ketchum KA, Kinsinger CR, Mesri M, Meyer MR, Qian WJ, Schoenherr RM, Scott MG, Shi T, Whiteley GR, Wrobel JA, Wu C, Ackermann BL, Aebersold R, Barnidge DR, Bunk DM, Clarke N, Fishman JB, Grant RP, Kusebauch U, Kushnir MM, Lowenthal MS, Moritz RL, Neubert H, Patterson SD, Rockwood AL, Rogers J, Singh RJ, Van Eyk JE, Wong SH, Zhang S, Chan DW, Chen X, Ellis MJ, Liebler DC, Rodland KD, Rodriguez H, Smith RD, Zhang Z, Zhang H, and Paulovich AG
- Subjects
- Guidelines as Topic, Humans, Peptides isolation & purification, Research Personnel, Clinical Laboratory Techniques, Mass Spectrometry, Peptides analysis, Proteomics, Specimen Handling
- Abstract
Background: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays., Content: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care., (© 2015 American Association for Clinical Chemistry.)
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- 2016
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20. Clonotypic Light Chain Peptides Identified for Monitoring Minimal Residual Disease in Multiple Myeloma without Bone Marrow Aspiration.
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Bergen HR 3rd, Dasari S, Dispenzieri A, Mills JR, Ramirez-Alvarado M, Tschumper RC, Jelinek DF, Barnidge DR, and Murray DL
- Subjects
- Bone Marrow pathology, Bone Marrow Examination, Complementarity Determining Regions blood, Complementarity Determining Regions genetics, Humans, Immunoglobulin Light Chains genetics, Multiple Myeloma pathology, Neoplasm, Residual pathology, Peptides genetics, RNA, Messenger blood, RNA, Messenger genetics, Suction, Immunoglobulin Light Chains blood, Multiple Myeloma blood, Multiple Myeloma diagnosis, Neoplasm, Residual blood, Neoplasm, Residual diagnosis, Peptides blood
- Abstract
Background: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions., Methods: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment., Results: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%)., Conclusions: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration., (© 2015 American Association for Clinical Chemistry.)
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- 2016
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21. Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.
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Barnidge DR, Lundström SL, Zhang B, Dasari S, Murray DL, and Zubarev RA
- Subjects
- Amino Acid Sequence, Antibody Diversity, Humans, Immunoglobulin Variable Region blood, Immunoglobulin Variable Region chemistry, Immunoglobulin Variable Region genetics, Immunoglobulin kappa-Chains blood, Immunoglobulin kappa-Chains genetics, Immunoglobulin lambda-Chains blood, Immunoglobulin lambda-Chains genetics, Molecular Sequence Data, Molecular Weight, Phenotype, Sequence Alignment, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization methods, Immunoglobulin kappa-Chains chemistry, Immunoglobulin lambda-Chains chemistry
- Abstract
In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.
- Published
- 2015
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22. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect monoclonal immunoglobulin light chains in serum and urine.
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Barnidge DR, Krick TP, Griffin TJ, and Murray DL
- Subjects
- Humans, Paraproteinemias blood, Paraproteinemias diagnosis, Paraproteinemias urine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Antibodies, Monoclonal blood, Antibodies, Monoclonal urine, Immunoglobulin Light Chains blood, Immunoglobulin Light Chains urine, Tandem Mass Spectrometry methods
- Abstract
Rationale: Use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to monitor serum and urine samples for endogenous monoclonal immunoglobulins. MALDI-TOFMS is faster, fully automatable, and provides superior specificity compared to protein gel electrophoresis (PEL)., Methods: Samples were enriched for immunoglobulins in 5 min using Melon Gel™ followed by reduction with dithiothreitol for 15 min to separate immunoglobulin light chains and heavy chains. Samples were then desalted using C4 ZipTips, mixed with sinapinic acid matrix, and analyzed on a Bruker Biflex III MALDI-TOF mass spectrometer., Results: Monoclonal immunoglobulin light chains were identified in serum and urine samples from patients with a known monoclonal gammopathy using MALDI-TOFMS with minimal sample preparation., Conclusions: MALDI-TOFMS can identify a monoclonal immunoglobulin in serum and urine samples. The molecular mass of the monoclonal immunoglobulin light chain is obtained providing unprecedented specificity compared to PEL. In addition, the methodology can be automated, making it a practical alternative to PEL., (Copyright © 2015 John Wiley & Sons, Ltd.)
- Published
- 2015
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23. Monitoring oligoclonal immunoglobulins in cerebral spinal fluid using microLC-ESI-Q-TOF mass spectrometry.
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Barnidge DR, Kohlhagen MC, Zheng S, Willrich MA, Katzmann JA, Pittock SJ, and Murray DL
- Subjects
- Chromatography, Liquid methods, Humans, Mass Spectrometry methods, Oligoclonal Bands cerebrospinal fluid
- Abstract
Our group has previously shown that mass spectrometry can be used to detect and quantify monoclonal and polyclonal immunoglobulin light chains in serum and urine from patients with monoclonal gammopathies and polyclonal hypergammaglobinemia. Here we demonstrate the use of the methodology, also referred to as monoclonal immunoglobulin Rapid Accurate Mass Measurement\with (miRAMM), to detect oligoclonal immunoglobulins above the polyclonal background in cerebral spinal fluid (CSF) and serum. We compared the findings for 56 paired CSF and serum samples analyzed by IgG IEF and miRAMM. The two methods were in agreement with 54 samples having concordant results (22 positive and 34 negative) and 2 that were positive by IgG IEF but negative by miRAMM. In addition to identifying oligoclonal immunoglobulins, miRAMM can be used to quantify and isotype each specific monoclonal immunoglobulin in CSF. This methodology has the potential to transform the way the inflammatory response is monitored in the CNS compartment., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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24. Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS.
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Willrich MA, Murray DL, Barnidge DR, Ladwig PM, and Snyder MR
- Subjects
- Chromatography, Liquid, Humans, Immunoglobulin G, Peptides, Tandem Mass Spectrometry, Anti-Inflammatory Agents blood, Antirheumatic Agents blood, Infliximab blood
- Abstract
Objectives: Although therapeutic concentrations of infliximab (chimeric IgG1 kappa) are associated with improved clinical prognosis, clinical laboratory methods for monitoring are limited. Therefore, we aimed to develop a LC-MS/MS method to measure infliximab in serum., Methods: Infliximab was measured using isotope-labeled peptides and horse IgG as a surrogate internal standard. After trypsin digestion, peptides were separated by reverse-phase C8 LC and detected by MS/MS on an ABSciex API 5000; analyte-to-internal standard peak area ratios were used for quantitation. Sera from patients receiving infliximab were collected at different time points in treatment and compared with a commercially available ECLIA method., Results: The linear dynamic range of the assay was 1-100 μg/mL (R(2)>0.998); both intra- and inter-assay imprecisions were <20%. Patients undergoing infliximab therapy had trough concentrations of 8.5 ± 8.8 μg/mL (mean ± SD), which substantially increased 48-72 h after infusion (77 ± 40 μg/mL), then fell after 28-32 days (15 ± 11 μg/mL). A comparison of LC-MS/MS and ECLIA methods demonstrated a slope of 0.967 (95% CI: 0.894-1.034, r=0.970)., Conclusions: We have demonstrated the ability to quantify infliximab in patients using clonotypic peptides. This approach has the potential to be quickly adaptable to other monoclonal antibodies and to expand the availability of testing for this class of therapeutics in routine clinical practice., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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25. Detecting monoclonal immunoglobulins in human serum using mass spectrometry.
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Mills JR, Barnidge DR, and Murray DL
- Subjects
- Antibodies, Monoclonal immunology, Chromatography, Liquid methods, Humans, Immunoglobulin Light Chains blood, Immunoglobulin Light Chains immunology, Myeloma Proteins immunology, Paraproteinemias immunology, Antibodies, Monoclonal blood, Mass Spectrometry methods, Myeloma Proteins analysis, Paraproteinemias diagnosis
- Abstract
Established guidelines from the International Myeloma Working Group recommend diagnostic screening for patients suspected of plasma cell proliferative disease using protein electrophoresis (PEL), free light chain measurements and immunofixation electrophoresis (IFE) of serum and urine in certain cases. Plasma cell proliferative disorders are generally classified as monoclonal gammopathies given most are associated with the excess secretion of a monoclonal immunoglobulin or M-protein. In clinical practice, the M-protein is detected in a patients' serum by the appearance of a distinct protein band migrating within regions typically occupied by immunoglobulins. Given each M-protein is comprised by a sequence of amino acids pre-defined by somatic recombination unique to each clonal plasma cell, the molecular mass of the M-protein can act as a surrogate marker. We established a mass spectrometry based method to assign molecular mass to the immunoglobulin light chain of the M-protein and used this to detect the presence of M-proteins. Our method first enriches serum for immunoglobulins, followed by reduction to separate light chains from heavy chains, followed by microflow LC-ESI-Q-TOF MS. The multiply charged light chain ions are converted to their molecular mass and reconstructed peak area calculations are used for quantification. Using this method, we term "monoclonal immunoglobulin Rapid Accurate Molecular Mass" or miRAMM, the presence of M-proteins can be reliably detected with superior sensitivity compared to current gel-based PEL and IFE techniques., (Copyright © 2015. Published by Elsevier Inc.)
- Published
- 2015
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26. Direct evidence of podocyte damage in cardiorenal syndrome type 2: preliminary evidence.
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Le Jemtel TH, Rajapreyar I, Selby MG, Payne B, Barnidge DR, Milic N, and Garovic VD
- Abstract
Background: Renal structural alterations have been partially uncovered in cardiorenal syndrome (CRS). Patients with CRS may have evidence of tubular damage, but markers of glomerular damage other than proteinuria have not been thoroughly investigated. The nature of renal damage in CRS may have therapeutic implications, as glomerular damage requires tight blood pressure control and renin-angiotensin-aldosterone system (RAAS) inhibition. The present investigation evaluates patients with CRS type 2 (CRS-2) for direct evidence of glomerular damage as evidenced by the presence of urinary podocin., Methods: The presence of glomerular damage was assessed in acutely decompensated patients with CRS-2 and healthy controls. Urinary podocin was determined by quantification of a tryptic peptide of podocin with high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Morning urine samples were collected for podocin, creatinine (Cr), and protein. Urinary podocin was expressed in femtomoles of podocin/milligram of Cr., Results: The urinary podocin/Cr ratio was greater in patients than in controls (0.37 ± 0.77 vs. 0.06 ± 0.05 fmol podocin/mg Cr, p = 0.04). A total of 40% of the patients had a urinary podocin/Cr ratio greater than the upper limit of normal (>0.2 fmol podocin/mg Cr). Patients with an elevated podocin/Cr ratio were more likely to have received ≤50% of the maximum dose of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (p = 0.04) than patients with a podocin/Cr ratio in the normal range., Conclusions: CRS-2 may be associated with glomerular damage as evidenced by an elevated urinary podocin/Cr ratio. Modulators of RAAS may have a protective effect on urinary podocin loss.
- Published
- 2015
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27. Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.
- Author
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Barnidge DR, Dasari S, Ramirez-Alvarado M, Fontan A, Willrich MA, Tschumper RC, Jelinek DF, Snyder MR, Dispenzieri A, Katzmann JA, and Murray DL
- Subjects
- Case-Control Studies, Humans, Immunoglobulin Light Chains blood, Immunoglobulin lambda-Chains blood, Immunoglobulin lambda-Chains chemistry, Linear Models, Molecular Weight, Phenotype, Reference Values, Agammaglobulinemia blood, Hypergammaglobulinemia blood, Immunoglobulin G blood, Immunoglobulin G chemistry, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.
- Published
- 2014
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28. Detecting monoclonal light chains in urine: microLC-ESI-Q-TOF mass spectrometry compared to immunofixation electrophoresis.
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Botz CM, Barnidge DR, Murray DL, and Katzmann JA
- Subjects
- Humans, Immunoelectrophoresis, Microchemistry, Molecular Weight, Osmolar Concentration, Proteinuria immunology, Urinalysis economics, Urinalysis methods, Waldenstrom Macroglobulinemia urine, Antibodies, Monoclonal urine, Immunoglobulin Light Chains urine, Proteinuria urine, Spectrometry, Mass, Electrospray Ionization economics
- Published
- 2014
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29. Quantification of serum IgG subclasses by use of subclass-specific tryptic peptides and liquid chromatography--tandem mass spectrometry.
- Author
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Ladwig PM, Barnidge DR, Snyder MR, Katzmann JA, and Murray DL
- Subjects
- Humans, Immunoglobulin G chemistry, Reference Standards, Chromatography, Liquid methods, Immunoglobulin G blood, Peptide Mapping, Tandem Mass Spectrometry methods, Trypsin chemistry
- Abstract
Background: Measurement of IgG subclasses is a useful tool for investigation of humoral immune deficiency in the presence of total IgG within reference intervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We describe an LC-MS/MS method that can multiplex all 4 subclasses along with total IgG by use of either IgG subclass-specific peptide stable isotope-labeled internal standards or a surrogate digest standard for quantification and does not rely on antigen/antibody reactions., Methods: We combined serum with labeled internal peptide standards and intact purified horse IgG. Samples were denatured, reduced, alkylated, and digested. We analyzed the digested serum by LC-MS/MS for IgG subclasses 1-4 and total IgG., Results: We assayed 112 patient sera by LC-MS/MS and immunonephelometry. The mean of the slopes and R(2) values for IgG1, IgG2, IgG3, IgG4, and IgG were 1.18 and 0.93, respectively. Interassay imprecision for the LC-MS/MS method was <15% for total IgG and subclasses and was slightly improved by use of a calibrator peptide from an exogenous horse IgG. Summed total IgG correlated with the measured total IgG within 10%. Reference intervals and analytical measuring range were all similar to our previous validation data for the immunonephelometry assays., Conclusions: Total IgG and IgG subclasses 1, 2, 3, and 4 can be quantified by LC-MS/MS with performance comparable to nephelometry., (© 2014 American Association for Clinical Chemistry.)
- Published
- 2014
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30. Monitoring M-proteins in patients with multiple myeloma using heavy-chain variable region clonotypic peptides and LC-MS/MS.
- Author
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Barnidge DR, Tschumper RC, Theis JD, Snyder MR, Jelinek DF, Katzmann JA, Dispenzieri A, and Murray DL
- Subjects
- Chromatography, Liquid, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Peptides, Immunoglobulins blood, Immunoglobulins chemistry, Multiple Myeloma blood, Proteomics methods, Tandem Mass Spectrometry methods
- Abstract
Multiple myeloma is a disease characterized by a clonal expansion of plasma cells that secrete a monoclonal immunoglobulin also referred to as an M-protein. In the clinical laboratory, protein electrophoresis (PEL), immunofixation electrophoresis (IFE), and free light chain nephelometry (FLC) are used to detect, monitor, and quantify an M-protein. Here, we present an alternative method based on monitoring a clonotypic (i.e., clone-specific) peptide from the M-protein heavy chain variable region using LC-MS/MS. Tryptic digests were performed on IgG purified serum from 10 patients with a known IgG M-protein. Digests were analyzed by shotgun LC-MS/MS, and the results were searched against a protein database with the patient specific, heavy chain variable region gene sequence added to the database. In all 10 cases, the protein database search matched multiple clonotypic peptides from each patient's heavy chain variable region. The clonotypic peptides were then used to quantitate the amount of M-protein in patient serum samples using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The response for the clonotypic peptide observed by SRM correlated with the M-protein observed by PEL. In addition, the clonotypic peptide was clearly observed by SRM in samples that were negative by IFE and FLC. Monitoring clonotypic peptides using SRM has the capacity to redefine clinical residual disease because of its superior sensitivity and specificity compared with current analytical methods.
- Published
- 2014
- Full Text
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31. Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy.
- Author
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Barnidge DR, Dasari S, Botz CM, Murray DH, Snyder MR, Katzmann JA, Dispenzieri A, and Murray DL
- Subjects
- Chromatography, Liquid methods, Humans, Multiple Myeloma diagnosis, Multiple Myeloma immunology, Paraproteinemias diagnosis, Paraproteinemias immunology, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Immunoglobulin Heavy Chains isolation & purification, Immunoglobulin Light Chains isolation & purification, Multiple Myeloma blood, Myeloma Proteins isolation & purification, Paraproteinemias blood
- Abstract
A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.
- Published
- 2014
- Full Text
- View/download PDF
32. Mass spectrometry as a novel method for detection of podocyturia in pre-eclampsia.
- Author
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Garovic VD, Craici IM, Wagner SJ, White WM, Brost BC, Rose CH, Grande JP, and Barnidge DR
- Subjects
- Adult, Chromatography, Liquid, Female, HELLP Syndrome urine, Humans, Podocytes chemistry, Pre-Eclampsia urine, Pregnancy, Prognosis, Proteinuria urine, Biomarkers urine, HELLP Syndrome diagnosis, Podocytes pathology, Pre-Eclampsia diagnosis, Proteinuria diagnosis, Tandem Mass Spectrometry
- Abstract
Background: Podocyturia, i.e. urinary loss of viable podocytes, may serve as a diagnostic tool for pre-eclampsia and as a marker of active renal disease. The current method to detect podocyturia is technically complex, lengthy and requires a high level of expertise for interpretation. The aim of this study was to develop a new technique for the identification of urinary podocytes, based on the detection of podocyte-specific tryptic peptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), which will provide an operator-independent and highly reproducible method., Methods and Results: The diagnosis of pre-eclampsia was confirmed in the presence of hypertension (>140/90 mmHg) and proteinuria >0.3 g/24 h urine. The diagnosis of HELLP was confirmed based on the accepted clinical criteria of hemolysis, elevated liver enzymes and low platelet count. Random urine samples within 24 h prior to delivery were collected and centrifuged. One half of the sediment was cultured for 24 h to select for viable cells and then stained with a podocin antibody, followed by a secondary fluorescein isothiocyanate-labeled antibody to identify podocytes. The second half of the pellet was solubilized, digested and analyzed by LC-MS/MS using an internal standard. We have recruited 13 patients with pre-eclampsia and 6 patients with pre-eclampsia/HELLP syndrome. The presence of podocytes was confirmed in all patients by the podocyte culture method. In the respective samples, the presence of a podocin-specific tryptic peptide was confirmed with LC-MS/MS technology., Conclusion: The LC-MS/MS method is a reliable technology for the identification of urinary podocytes, based on the presence of podocyte-specific proteins in the urine.
- Published
- 2013
- Full Text
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33. Washing mineral oil reduces contaminants and embryotoxicity.
- Author
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Morbeck DE, Khan Z, Barnidge DR, and Walker DL
- Subjects
- Aldehydes analysis, Aldehydes pharmacology, Alkenes analysis, Alkenes pharmacology, Animals, Culture Media toxicity, Detergents pharmacology, Embryo, Mammalian physiology, Humans, Male, Mice, Mineral Oil pharmacology, Octoxynol pharmacology, Peroxides analysis, Peroxides pharmacology, Semen Analysis, Spermatozoa drug effects, Spermatozoa physiology, Toxins, Biological analysis, Toxins, Biological isolation & purification, Toxins, Biological toxicity, Water pharmacology, Drug Contamination, Embryo Culture Techniques methods, Embryo, Mammalian drug effects, Mineral Oil toxicity
- Abstract
Objective: To determine if washing improves the quality of mineral oil used for embryo culture., Design: A 2 × 3 factorial experimental study., Setting: University hospital-based infertility center., Animal(s): Mice., Intervention(s): The chemical nature of contaminants present in two lots of mineral oil was determined. Effect of washing on toxicity and amount of toxin present in media was determined., Main Outcome Measure(s): The effect of washing was determined by a quality control bioassay or by directly determining the level of contaminant in oil-conditioned culture media., Result(s): Water, culture media, and media plus albumin were equally effective in reducing toxicity and concentration of toxin. Temperature did not affect washing results. Peroxide, aldehydes, and alkenals were present in one lot of oil, and Triton X-100 was identified in the other lot. Washed oil containing peroxide passed the one-cell mouse embryo bioassay, and washing reduced the amount of Triton X-100 by 25%., Conclusion(s): Mineral oil is the least defined component used for in vitro fertilization and embryo culture; therefore, it is important to determine if washing oil is beneficial. This study provides clear evidence that washing reduces toxicity of mineral oil., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
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34. Tools for building clinic-community partnerships to support chronic disease control and prevention.
- Author
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Barnidge EK, Brownson CA, Baker EA, and Shetty G
- Subjects
- Diabetes Mellitus rehabilitation, Humans, Leadership, Patient Care Planning, Chronic Disease prevention & control, Community-Institutional Relations, Patient Education as Topic, Self Care
- Abstract
Purpose: Developing partnerships among health care clinics and community organizations is an important strategy for increasing resources and supports for chronic disease care and management. Although several tools assessing partnership characteristics exist, tools to assess the progression from partnership development to the achievement of specific short-term, intermediate, and long-term outcomes have not been developed to date. The purpose of this article is to introduce tools developed by the Diabetes Initiative of the Robert Wood Johnson Foundation to fill that gap., Conclusion: The Diabetes Initiative used a group process with program grantees to better delineate the phases of partnership development that contribute to the achievement of a shared long-term goal. The Framework for Building Clinic-Community Partnerships to Support Chronic Disease Control and Prevention presented in this article was developed as a result of this process. To apply the framework, 3 checklists were created to correspond to each stage of the framework. The final tools include the framework; 3 checklists with items to assess partnership development, agency capacity within and between agencies, and intermediate and long-term outcomes; and a form to facilitate changes to improve the partnership. Overall, these tools seek to aid partnerships in achieving the best possible chronic disease outcomes.
- Published
- 2010
- Full Text
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35. Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.
- Author
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Kumar V, Barnidge DR, Chen LS, Twentyman JM, Cradic KW, Grebe SK, and Singh RJ
- Subjects
- Humans, Hyperparathyroidism diagnosis, Limit of Detection, Linear Models, Reproducibility of Results, Chromatography, Liquid methods, Hyperparathyroidism blood, Immunoassay methods, Parathyroid Hormone blood, Tandem Mass Spectrometry methods
- Abstract
Background: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues., Methods: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH ((1)SVSEIQLMHNLGK(13))., Results: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was
- Published
- 2010
- Full Text
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36. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants.
- Author
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Lopez MF, Rezai T, Sarracino DA, Prakash A, Krastins B, Athanas M, Singh RJ, Barnidge DR, Oran P, Borges C, and Nelson RW
- Subjects
- Aged, Amino Acid Sequence, Area Under Curve, Female, Humans, Kidney Failure, Chronic blood, Limit of Detection, Male, Molecular Sequence Data, Immunoassay methods, Kidney Failure, Chronic diagnosis, Parathyroid Hormone blood, Peptide Fragments blood, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
Background: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84., Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards., Results: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R(2) = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%., Conclusions: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.
- Published
- 2010
- Full Text
- View/download PDF
37. Quantification of urinary albumin by using protein cleavage and LC-MS/MS.
- Author
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Seegmiller JC, Barnidge DR, Burns BE, Larson TS, Lieske JC, and Kumar R
- Subjects
- Albumins chemistry, Albuminuria urine, Amino Acid Sequence, Calibration, Humans, Hydrolysis, Reproducibility of Results, Sensitivity and Specificity, Albumins analysis, Albuminuria diagnosis, Chromatography, Liquid methods, Tandem Mass Spectrometry methods
- Abstract
Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ)., Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, (15)N-labeled recombinant HSA ((15)NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides (42)LVNEVTEFAK(51) and (526)QTALVELVK(534) from albumin and (15)NrHSA were referenced to the calibration curve., Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide (42)LVNEVTEFAK(51), and 1.9%-12.3% for the (526)QTALVELVK(534) peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the (42)LVNEVTEFAK(51) peptide, and 6.4%-14.1% for the (526)QTALVELVK(534) peptide. For the (42)LVNEVTEFAK(51) peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R(2) value of 0.97 and a regression line of y = 0.99x + 23.16., Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a (15)NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.
- Published
- 2009
- Full Text
- View/download PDF
38. LC-MS/MS quantification of Zn-alpha2 glycoprotein: a potential serum biomarker for prostate cancer.
- Author
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Bondar OP, Barnidge DR, Klee EW, Davis BJ, and Klee GG
- Subjects
- Adipokines, Chromatography, Liquid, Humans, Hydrolysis, Male, Peptides blood, Pilot Projects, Reproducibility of Results, Sensitivity and Specificity, Serum, Tandem Mass Spectrometry, Biomarkers, Tumor blood, Carrier Proteins blood, Glycoproteins blood, Prostatic Neoplasms diagnosis
- Abstract
Background: Zn-alpha2 glycoprotein (ZAG) is a relatively abundant glycoprotein that has potential as a biomarker for prostate cancer. We present a high-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring serum ZAG concentrations by proteolytic cleavage of the protein and quantification of a unique peptide., Methods: We selected the ZAG tryptic peptide (147)EIPAWVPEDPAAQITK(162) as the intact protein for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Standards using recombinant ZAG in bovine serum albumin, 50 g/L, and a pilot series of patient sera were denatured, reduced, alkylated, and digested with trypsin. The concentration of ZAG was calculated from a dose-response curve of the ratio of the relative abundance of the ZAG tryptic peptide to internal standard., Results: The limit of detection for ZAG in serum was 0.08 mg/L, and the limit of quantification was 0.32 mg/L with a linear dynamic range of 0.32 to 10.2 mg/L. Replicate digests from pooled sera run during a period of 3 consecutive days showed intraassay imprecision (CV) of 5.0% to 6.3% and interassay imprecision of 4.4% to 5.9%. Mean (SD) ZAG was higher in 25 men with prostate cancer [7.59 (2.45) mg/L] than in 20 men with nonmalignant prostate disease [6.21 (1.65) mg/L, P = 0.037] and 6 healthy men [3.65 (0.71) mg/L, P = 0.0007]., Conclusions: This LC-MS/MS assay is reproducible and can be used to evaluate the clinical utility of ZAG as a cancer biomarker.
- Published
- 2007
- Full Text
- View/download PDF
39. Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.
- Author
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Barnidge DR, Jelinek DF, Muddiman DC, and Kay NE
- Subjects
- Cell Fractionation, Humans, Immunoglobulin Heavy Chains immunology, Immunoglobulin Variable Region immunology, Mass Spectrometry methods, Mutation, B-Lymphocytes chemistry, B-Lymphocytes immunology, Genes, Immunoglobulin, Immunoglobulin Heavy Chains genetics, Immunoglobulin Variable Region genetics, Isotope Labeling methods, Leukemia, Lymphocytic, Chronic, B-Cell immunology, Proteome analysis
- Abstract
Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.
- Published
- 2005
- Full Text
- View/download PDF
40. Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC-MS/MS.
- Author
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Barnidge DR, Tschumper RC, Jelinek DF, Muddiman DC, and Kay NE
- Subjects
- Antigens, CD isolation & purification, Blood Proteins isolation & purification, Cell Fractionation, Cell Membrane chemistry, Humans, Neoplasm Proteins blood, B-Lymphocytes metabolism, Chromatography, Ion Exchange methods, Chromatography, Liquid methods, Gene Expression Profiling methods, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Mass Spectrometry methods
- Abstract
In this study we use replicate 2D-LC-MS/MS analyses of crude membranes from B cells derived from a patient with chronic lymphocytic leukemia (CLL) to examine the protein expression profile of CLL B cells. Protein identifications made by replicate 2D-LC-MS/MS analysis of tryptic peptides from detergent solubilized B cell membrane proteins, as well as replicate LC-MS/MS analysis of single off-line strong cation exchange chromatography (SCX) fractions, were analyzed. We show that despite the variance in SCX, capillary LC, and the data-dependent selection of precursor ions, an overlap of 64% between proteins identified in replicate runs was achieved for this system.
- Published
- 2005
- Full Text
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41. Evaluation of a cleavable stable isotope labeled synthetic peptide for absolute protein quantification using LC-MS/MS.
- Author
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Barnidge DR, Hall GD, Stocker JL, and Muddiman DC
- Subjects
- Chromatography, Liquid, Isotope Labeling, Mass Spectrometry, Peptides chemistry
- Abstract
Protein cleavage coupled with isotope dilution mass spectrometry (PC-IDMS) has the potential to provide the absolute concentration of a specific protein, or multiple proteins, in complex mixtures. However, PC-IDMS differs from standard IDMS since the internal standard is a different molecule than the analyte at the start of the experiment, more specifically, the internal standard is a peptide and the analyte is a protein prior to cleavage. It is not until after the cleavage process that the stable isotope labeled synthetic peptide has the same physicochemical behavior as the peptide cleaved from the protein. The work presented here evaluates the use of tryptic cleavage sites incorporated into the internal standard synthetic peptide in an attempt to create an internal standard that has cleavage characteristics more similar to the protein being quantified. Results presented here suggest that an internal standard synthetic peptide incorporating internal cleavage sites does not improve the accuracy and precision of the values obtained when performing PC-IDMS.
- Published
- 2004
- Full Text
- View/download PDF
42. Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry.
- Author
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Barnidge DR, Goodmanson MK, Klee GG, and Muddiman DC
- Subjects
- Humans, Male, Mass Spectrometry, Protein Processing, Post-Translational, Trypsin chemistry, Urea chemistry, Isotope Labeling, Peptide Fragments chemistry, Prostate-Specific Antigen chemistry, Serum chemistry
- Abstract
Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.
- Published
- 2004
- Full Text
- View/download PDF
43. Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.
- Author
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Barnidge DR, Dratz EA, Martin T, Bonilla LE, Moran LB, and Lindall A
- Subjects
- Chromatography, Liquid methods, Peptide Fragments chemistry, Peptides chemical synthesis, Peptides standards, Reference Standards, Spectrometry, Mass, Electrospray Ionization methods, GTP-Binding Proteins metabolism, Rhodopsin analysis
- Abstract
Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.
- Published
- 2003
- Full Text
- View/download PDF
44. A design for low-flow sheathless electrospray emitters.
- Author
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Barnidge DR, Nilsson S, and Markides KE
- Abstract
An extremely simple design has been developed for producing durable sheathless electrospray emitters that give highly stable electrospray for unlimited lifetimes. The emitters can be fashioned from any style fused-silica capillary and are ideally suited for generating "all-in-one" microcolumn-emitter systems thus eliminating unwanted void volumes. The emitters give stable electrospray at low (30 nL/min) as well as high (1 mL/min) flow rates without the aid of nebulizing gas. Fabrication of these emitters (aka the "fairy dust" technique) does not involve the use of a metallized coating but rather the adherance of 2-μm gold particles to the capillary tip resulting in a robust approach to the problem of making an electrical contact with the electrospray solvent.
- Published
- 1999
- Full Text
- View/download PDF
45. Extraction method for analysis of detergent-solubilized bacteriorhodopsin and hydrophobic peptides by electrospray ionization mass spectrometry.
- Author
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Barnidge DR, Dratz EA, Jesaitis AJ, and Sunner J
- Subjects
- Bacteriorhodopsins chemistry, Cell Extracts chemistry, Detergents chemistry, Halobacterium salinarum metabolism, Membrane Proteins analysis, Peptides chemistry, Solubility, Solutions, Bacteriorhodopsins analysis, Mass Spectrometry methods, Peptides analysis
- Abstract
The analysis of integral membrane proteins or transmembrane peptides by electrospray ionization mass spectrometry (ESI-MS) is difficult since detergents, used to solubilize these hydrophobic proteins and peptides, severely suppress analyte ion formation. This problem has been addressed previously by precipitating the protein, removing the detergent, and resolubilizing the protein in a nonpolar solvent. Here, we demonstrate a method that avoids protein precipitation and resolubilization. Detergent-solubilized bacteriorhodopsin is extracted into a nonpolar solvent phase by adding a chloroform/methanol/water solvent mixture to the aqueous detergent solution. ESI mass spectra of the nonpolar, chloroform-rich phase were dominated by peaks due to bacterioopsin. Bacterioopsin precursors with partially cleaved leader sequences were seen in all mass spectra. Additional peaks were likely due to intact bacteriorhodopsin, i.e., bacterioopsin with the retinal prosthetic group attached, and to bacterioopsin associated with lipid molecules. A separation process that occurred in the fused-silica capillary leading to the electrospray tip was essential for obtaining ESI mass spectra of bacterioopsin. The extraction-into-chloroform procedure also worked well with hydrophobic, transmembrane-type peptides that were insoluble in other electrospray solvents, including 100% formic acid, and the method has application to transmembrane peptides formed from digests of integral membrane proteins., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
46. Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
- Author
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Barnidge DR, Dratz EA, Sunner J, and Jesaitis AJ
- Subjects
- Amino Acid Sequence, Animals, Cattle, Glycosylation, Molecular Sequence Data, Protein Conformation, Rod Cell Outer Segment chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Membrane Proteins chemistry, Peptide Fragments chemistry, Rhodopsin chemistry, Trypsin chemistry
- Abstract
The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ability to determine the accurate mass of hydrophobic transmembrane peptides will facilitate the mapping of ligand binding sites in membrane receptors. It will also augment the determination of membrane spanning regions from integral membrane proteins digested in lipid bilayers. Affinity-purified rhodopsin in detergent and rhodopsin in retinal rod membranes were digested with trypsin. Tryptic peptides were separated using reverse-phase, high-performance liquid chromatography at 55 degrees C with the detergent octyl-beta-glucoside in the mobile phase. Four of the six transmembrane tryptic peptides of rhodopsin were identified, ranging in mass from 3,260 Da to 6,528 Da. The identities of the peptides were confirmed by Edman microsequencing. In addition, heterogeneity in the glycosylation of the N-terminal tryptic peptide of rhodopsin was identified by MALDI MS, without modifying the carbohydrate prior to analysis.
- Published
- 1997
- Full Text
- View/download PDF
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