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The use of mass spectrometry methods with triple quadrupole instruments is well established for quantification. However, the preparation of calibration curves can be time-consuming and prone to analytical errors. In this study, an innovative internal calibration (IC) approach using a one-standard calibration with a stable isotope-labeled (SIL) standard version of the endogenous compound was developed. To ensure optimal quantitative performance, the following parameters were evaluated: the stability of the analyte-to-SIL response factor (RF), the chemical and isotopic purities of the SIL, and the instrumental reproducibility. Using six clinically important endogenous steroids and their respective SIL standards, we demonstrated that RFs obtained on different LC-MS platforms were consistent. The quantitative performance of the proposed approach was determined using quality control samples prepared in depleted serum, and showed both satisfactory precision (1.3%-12.4%) and trueness (77.5%-107.0%, with only 3 values outside ±30%). The developed method was then applied to human serum samples, and the results were similar to those obtained with the conventional quantification approach based on external calibration: the Passing-Bablok regression showed a proportional bias of 6.8% and a mean difference of -5.9% between the two methodologies. Finally, we showed that the naturally occurring isotopes of the SIL can be used to provide additional calibration points and increase the accuracy for analytes with low concentrations.

Background Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. Methods We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. Results The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. Conclusions This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

The search for novel and clinically relevant biomarkers still represents a major clinical challenge and mass-spectrometry-based technologies are essential tools to help in this process. In this application, we demonstrate how selected reaction monitoring (SRM) can be applied in a highly multiplexed way to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Such an assay can be used to analyze numerous samples for narrowing down a list of potential biomarkers to the most relevant candidates. The use of FFPE tissues is of high relevance in this context as large sample collections linked with valuable clinical information are available in hospitals around the world. Here we describe in detail how we proceeded to develop such an assay for 200 proteins in breast tumor FFPE tissues. We cover the selection of suitable peptides, which are different in FFPE compared to fresh frozen tissues and show how we deliberately biased our assay toward proteins with a high probability of being measurable in human clinical samples.

The prevalence of chronic kidney disease (CKD) is increasing worldwide. New technical approaches are needed to improve early diagnosis, disease understanding and patient monitoring, and to evaluate new therapies. Metabolomics, as a prime candidate in the field of CKD research, aims to comprehensively analyze the metabolic complexity of biological systems. An extensive analysis of the metabolites contained in biofluids is therefore needed, and the combination of data obtained from multiple analytical platforms constitutes a promising methodological approach. This study presents an original workflow based on complementary chromatographic conditions, reversed-phase and hydrophilic interaction chromatography hyphenated to mass spectrometry to improve the polar metabolome coverage coupled with a univocal metabolite annotation strategy enabling a rapid access to the biological interpretation. This multiplatform workflow was applied in a CKD cohort study to assess plasma metabolic profile modifications related to renal disease. Multivariate analysis of 278 endogenous annotated metabolites enabled patient stratification with respect to CKD stages and helped to generate new biological insights, while also confirming the relevance of tryptophan metabolism pathway in this condition.

Clinical mass spectrometry proteomics (cMSP) assays are being increasingly used in clinical laboratories for analyzing peptides and proteins. It has therefore become urgent to characterize and validate the methods available for liquid chromatography-tandem mass spectrometry (LC/MS-MS) targeted quantification of peptide and protein biomarkers in biological fluids in the context of in vitro diagnostics. LC-MS/MS for the detection of peptides and proteins is currently the main approach used in the field of cMSP. As a result of their selectivity, low reagent costs and the fact that these methods can be used for absolute quantification and multiplexing, they will likely eventually replace immunoassays. Although LC-MS/MS is known to be the main reference method involved in reference measurement procedures (RMPs), it needs to meet the requirements of in vitro diagnostic (IVD) regulations and standards. This review shows that cMSP is fully compatible with the regulatory IVD requirements and provides an overview of the characterization and validation of the use of LC-MS/MS targeted quantification of clinical protein biomarkers in biological fluids.

Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin.Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides.Five structures of busulfan adducts were detected and a chemical structure could be attributed to four of them. Two were primary adducts corresponding to busulfan monoalkylation and alkylation of two amino acid residues by a single busulfan molecule. Two others corresponded to secondary adducts generated during sample processing. Adducts were mainly detected on Asp, Glu, and His residues. These findings were confirmed by subsequent database searches and experiments with synthetic peptides.The combination of in vitro incubation of proteins with the drug of interest or control compounds, high-resolution mass spectrometry, and open modification search allowed confirmation of the direct interaction of busulfan with proteins and characterization of the resulting adducts. Our results also showed that careful analysis of the data is required to detect experimental artifacts. Copyright © 2016 John WileySons, Ltd.

Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4°C, -20°C, and -80°C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80°C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20°C or -80°C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selected reaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R2: 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performances and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Objective To determine the prognostic accuracy of cardiac biomarkers alone and in combination with clinical scores in elderly patients with non-high-risk pulmonary embolism (PE). Design Ancillary analysis of a Swiss multicentre prospective cohort study. Subjects A total of 230 patients aged ≥65 years with non-high-risk PE. Main outcome measures The study end-point was a composite of PE-related complications, defined as PE-related death, recurrent venous thromboembolism or major bleeding during a follow-up of 30 days. The prognostic accuracy of the Pulmonary Embolism Severity Index (PESI), the Geneva Prognostic Score (GPS), the precursor of brain natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) was determined using sensitivity, specificity, predictive values, receiver operating characteristic (ROC) curve analysis, logistic regression and reclassification statistics. Results The overall complication rate during follow-up was 8.7%. hs-cTnT achieved the highest prognostic accuracy [area under the ROC curve: 0.75, 95% confidence interval (CI): 0.63–0.86, P

International audience; Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.

Cerebrospinal fluid (CSF) rhinorrhea is a serious condition that may result in severe complications. Various laboratory tests, relying on the detection of CSF-specific proteins in nasal secretions, have been developed but diagnosis remains challenging. The aim of this study was to evaluate two new methods targeting either ß2-transferrin or beta-trace-protein. Rhinorrhea samples from patients suspected of CSF leakage (n=36) were analyzed using two-dimensional gel electrophoresis (2-DE) for CSF rhinorrhea diagnosis. Twelve patients with rhinorrhea strongly suggestive of a CSF leak also underwent a fluorescein test. The same cohort was retrospectively analyzed with a beta-trace protein immunoblot developed in-house (n=36) and a new commercial ß2-transferrin immunofixation assay (Sebia, Evry, France) (n=33). 2-DE was positive in 9 patients suffering from rhinorrhea following skull base fracture (n=3), post-surgery (n=4), or spontaneously (n=2). The 27 remaining cases were negative. These results were confirmed by the beta-trace protein immunoblot and ß2-transferrin immunofixation tests, except for one sample found negative with 2-DE but positive with the two other assays. Results from the three analytical methods were concordant with fluorescein tests. Beta-trace protein immunoblot and ß2-transferrin immunofixation assays are fast and reliable methods that allow detecting CSF leakage in nasal fluid with high sensitivity and specificity.

Chronic kidney disease (CKD) is becoming a worldwide public health problem. The identification of a specific set of early biomarkers for CKD is extremely relevant to progress in disease knowledge, improving diagnosis, treatment, or development, and monitoring efficacy of new drugs. As kidney fibrosis can be considered the common pathological way to end stage renal failure, independent of the initial renal insult, these biomarkers are therefore biomarkers of early tubulo-interstitial fibrosis. The availability of a specific set of biomarkers for CKD is the mandatory condition to create new dedicated drugs and validate them in clinics without waiting years for a functional response in patients. We suggest here specific cohorts of patients where this early signature of fibrosis may be simpler to be identified.

Proteomics has stimulated the development of very powerful methods for protein analysis. Implementation of some of these methods in clinical chemistry laboratories could offer clinicians better tools for diagnosis, prognosis and therapeutic follow-up of human diseases. However, laboratory medicine activities are bound by a number of constraints and rules for ensuring quality of results for clinical practice. There is therefore a gap to be filled between the research and routine medical laboratories. In this opinion article, we present the proteomic methods that will most likely be implemented in clinical chemistry laboratories in the short term, and we discuss the major issues yet to be addressed before considering such a transfer.

Stenosis of the common bile duct may be either due to benign (chronic pancreatitis) or malignant (cholangiocarcinoma, pancreatic adenocarcinoma) conditions. The benign nature of the stricture should be first confirmed in order to ensure appropriate therapy. Therefore, the identification of markers allowing discrimination between malignant and benign biliary stenosis would be very valuable in clinical practice. To this intent, we performed a proteomic analysis of bile samples from patients having a biliary stenosis caused by pancreatic adenocarcinoma. Bile samples were collected during endoscopic retrograde cholangiopancreatography and purified using different methods. The extracted proteins were then analyzed by SDS-PAGE and LC-MS/MS. A total of 127 proteins were identified, 34 of which have not been previously detected in proteomic studies of bile. Among them, several proteins have been described as potential biomarkers of pancreatic cancer. We extended our investigation by studying the expression of some of these pancreatic cancer markers in bile samples collected from patients with various etiologies of biliary stenosis including pancreatic cancer, cholangiocarcinoma, chronic pancreatitis, as well as gallstone-induced stenosis. Our data showed a conspicuous overexpression of CEACAM6 and MUC1 (CA19-9) in pancreatic cancer and cholangiocarcinoma samples, according to the hypothesis that bile fluid collects cancer-associated protein leaking from the tumor microenvironment. These results underline the interest of using bile as a source of biomarkers for the diagnosis of malignant biliary stenosis.

The incidence of acute pancreatitis, an inflammation of the pancreas, is increasing worldwide. Pancreatic injury is mild in 80%-90% of patients who recover without complications. The remaining patients may develop a severe disease with local complications such as acinar cell necrosis, abscess and remote organ injury including lung injury. The early prediction of the severity of the disease is an important goal for physicians in management of patients with acute pancreatitis in order to optimize the therapy and to prevent organ dysfunction and local complications. For that purpose, multiple clinical scale scores have been applied to patients with acute pancreatitis. Recently, a new problem has emerged: the increased severity of the disease in obese patients. However, the mechanisms by which obesity increases the severity of acute pancreatitis are unclear. Several hypotheses have been suggested: (1) obese patients have an increased inflammation within the pancreas; (2) obese patients have an increased accumulation of fat within and around the pancreas where necrosis is often located; (3) increase in both peri- and intra-pancreatic fat and inflammatory cells explain the high incidence of pancreatic inflammation and necrosis in obese patients; (4) hepatic dysfunction associated with obesity might enhance the systemic inflammatory response by altering the detoxification of inflammatory mediators; and (5) ventilation/perfusion mismatch leading to hypoxia associated with a low pancreatic flow might reduce the pancreatic oxygenation and further enhance pancreatic injury. Recent experimental investigations also show an increased mortality and morbidity in obese rodents with acute pancreatitis and the implication of the adipokines leptin and adiponectin. Such models are important to investigate whether the inflammatory response of the disease is enhanced by obesity. It is exciting to speculate that manipulation of the adipokine milieu has the potential to influence the severity of acute pancreatitis.

Biomarker discovery for clinical purposes is one of the major areas in which proteomics is used. However, despite considerable effort, the successes have been relatively scarce. In this perspective paper, we try to highlight and analyze the main causes for this limited success, and to suggest alternate strategies, which will avoid them, without eluding the foreseeable weak points of these strategies. Two major strategies are analyzed, namely, the switch from body fluids to cell and tissues for the initial biomarker discovery step or, if body fluids must be analyzed, the implementation of highly selective protein selection strategies.

Proteomic technologies, such as yeast two-hybrid, mass spectrometry (MS), protein/peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories.

Heart-fatty acid-binding protein (H-FABP) is a small cytosolic protein involved in intracellular fatty acid transport. This protein, highly concentrated in the heart, is quickly released into plasma after myocardial injury. Results from numerous studies suggest that H-FABP is an excellent marker for the early detection of myocardial damage. H-FABP is also expressed in the brain, although in lower concentrations than in the heart. Recent preliminary studies also investigated the usefulness of H-FABP for the diagnosis of acute and chronic neurological disorders. These potential applications of H-FABP in clinical practice are reviewed in this article, with a strong focus on the early diagnosis of acute myocardial infarction and stroke.

A new, versatile, multiphasic buffer system for high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins in the relative molecular weight range of 300 000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above M(r) range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pK(a), as the buffering counterion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa M(r) range, with minimal difficulties in the post electrophoretic identification processes., website publisher http://www.interscience.wiley.com