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Background: The objective of this study was to develop a method for directly analysing osteochondral samples straight out of the operating room without cell culturing, thereby enabling identification of potential peptide biomarkers to better understand the mechanisms involved in the development of osteoarthritis and pain., Material and Methods: Osteochondral plugs from wounded and macroscopically nonwounded zones of the femur condyle were collected from six patients with manifest osteoarthritis (OA) undergoing total knee arthroplasty (TKA). The samples were demineralized and supernatant was collected and isotopically marked with Tandem Mass Tag (TMT) labelling and analysed using liquid chromatography coupled with tandem mass spectrometry LC-MS/MS., Results: Using peptidomics, 6292 endogenous peptides were identified. Five hundred sixty-six peptides (8 identified endogenous peptides) differed significantly (P-value 0.10) from wounded zones compared to nonwounded zones., Conclusion: This pilot study shows promising results for enabling peptidomic analysis of cartilage and bone straight out of the operating room. With further refinement, peptidomics can potentially become a diagnostic tool for OA, and improve the knowledge of disease progression and genesis of pain., (© 2019 Stichting European Society for Clinical Investigation Journal Foundation.)

We present a new, quantification-driven proteomic approach to identifying biomarkers. In contrast to the identification-driven approach, limited in scope to peptides that are identified by database searching in the first step, all MS data are considered to select biomarker candidates. The endopeptidome of cerebrospinal fluid from 40 Alzheimer's disease (AD) patients, 40 subjects with mild cognitive impairment, and 40 controls with subjective cognitive decline was analyzed using multiplex isobaric labeling. Spectral clustering was used to match MS/MS spectra. The top biomarker candidate cluster (215% higher in AD compared to controls, area under ROC curve = 0.96) was identified as a fragment of pleiotrophin located near the protein's C-terminus. Analysis of another cohort (n = 60 over four clinical groups) verified that the biomarker was increased in AD patients while no change in controls, Parkinson's disease or progressive supranuclear palsy was observed. The identification of the novel biomarker pleiotrophin 151-166 demonstrates that our quantification-driven proteomic approach is a promising method for biomarker discovery, which may be universally applicable in clinical proteomics.

Biomarkers of neurodegenerative disorders are needed to assist in diagnosis, to monitor disease progression and therapeutic interventions, and to provide insight into disease mechanisms. One route to identify such biomarkers is by proteomic and peptidomic analysis of cerebrospinal fluid (CSF). In the current study, we performed an in-depth analysis of the human CSF endopeptidome to establish an inventory that may serve as a basis for future targeted biomarker studies. High-pH RP HPLC was employed for off-line sample prefractionation followed by low-pH nano-LC-MS analysis. Different software programs and scoring algorithms for peptide identification were employed and compared. A total of 18 031 endogenous peptides were identified at a FDR of 1%, increasing the number of known endogenous CSF peptides 10-fold compared to previous studies. The peptides were derived from 2 053 proteins of which more than 60 have been linked to neurodegeneration. Notably, among the findings were six peptides derived from microtubule-associated protein tau, three of which span the diagnostically interesting threonine-181 (Tau-F isoform). Also, 213 peptides from amyloid precursor protein were identified, 58 of which were partially or completely within the sequence of amyloid β 1-40/42, as well as 109 peptides from apolipoprotein E, spanning sequences that discriminate between the E2/E3/E4 isoforms of the protein., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many disease processes in the brain are reflected in the protein composition of the cerebrospinal fluid (CSF). In addition to proteins, CSF also contains a large number of endogenous peptides whose potential as disease biomarkers largely remains to be explored. We have developed a novel workflow in which multiplex isobaric labeling is used for simultaneous quantification of endogenous CSF peptides and proteins by liquid chromatography coupled with mass spectrometry. After the labeling of CSF samples, endogenous peptides are separated from proteins by ultrafiltration. The proteins retained on the filters are trypsinized, and the tryptic peptides are collected separately. We evaluated this technique in a comparative pilot study of CSF peptide and protein profiles in eight patients with Alzheimer's disease (AD) and eight nondemented controls. We identified several differences between the AD and control group among endogenous peptides derived from proteins known to be associated with AD, including neurosecretory protein VGF (ratios AD/controls 0.45-0.81), integral membrane protein 2B (ratios AD/controls 0.72-0.84), and metallothionein-3 (ratios AD/controls 0.51-0.61). Analysis of tryptic peptides identified several proteins that were altered in the AD group, some of which have previously been reported as changed in AD, for example, VGF (ratio AD/controls 0.70).

We report on the analysis of endogenous peptides in cerebrospinal fluid (CSF) by mass spectrometry. A method was developed for preparation of peptide extracts from CSF. Analysis of the extracts by offline LC-MALDI MS resulted in the detection of 3,000-4,000 peptide-like features. Out of these, 730 peptides were identified by MS/MS. The majority of these peptides have not been previously reported in CSF. The identified peptides were found to originate from 104 proteins, of which several have been reported to be involved in different disorders of the central nervous system. These results support the notion that CSF peptidomics may be viable complement to proteomics in the search of biomarkers of CNS disorders.

Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture.

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.

The use of delayed ion extraction in MALDI time-of-flight mass spectrometry distorts the linear relationship between m/z and the square of the ion flight time (t2) with the consequence that, if a mass accuracy of 10 ppm or better is to be obtained, the calibrant signals have to fall close to the analyte signals. If this is not possible, systematic errors arise. To eliminate these, a higher-order calibration function and thus several calibrant signals are required. For internal calibration, however, this approach is limited by signal suppression effects and the increasing chance of the calibrant signals overlapping with analyte signals. If instead the calibrants are prepared separately, this problem is replaced by an other; i.e., the ion flight times are dependent on the sample plate position. For this reason, even if the calibrants are placed close to the sample, the mass accuracy is not improved when a higher-order calibration function is applied. We have studied this phenomenon and found that the relative errors, which result when moving from one sample to the next, are directly proportional to m/z. Based on this observation, we developed a two-step calibration method, that overcomes said limitations. The first step is an external calibration with a high-order polynomial function used for the determination of the relation between m/z and t2, and the second step is a first-order internal correction for sample position-dependent errors. Applying this method, for instance, to a mass spectrum of a mixture of 18 peptides from a tryptic digest of a recombinant protein resulted in an average mass error of 1.0 ppm with a standard deviation of 3.5 ppm. When instead using a conventional two-point internal calibration, the average relative error was 2.2 ppm with a standard deviation of 15 ppm. The new method is described and its performance is demonstrated with examples relevant to proteome research.

Brain amyloid‐β (Aβ) deposits are key pathological hallmarks of both cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). Microvascular deposits in CAA mainly consist of the Aβ40 peptide, whereas Aβ42 is the predominant variant in parenchymal plaques in AD. The relevance in pathogenesis and diagnostic accuracy of various other Aβ isoforms in CAA remain understudied. We aimed to investigate the biomarker potential of various Aβ isoforms in cerebrospinal fluid (CSF) to differentiate CAA from AD pathology. We included 25 patients with probable CAA, 50 subjects with a CSF profile indicative of AD pathology (AD‐like), and 23 age‐ and sex‐matched controls. CSF levels of Aβ1‐34, Aβ1‐37, Aβ1‐38, Aβ1‐39, Aβ1‐40, and Aβ1‐42 were quantified by liquid chromatography mass spectrometry. Lower CSF levels of all six Aβ peptides were observed in CAA patients compared with controls (p = 0.0005–0.03). Except for Aβ1‐42 (p = 1.0), all peptides were decreased in CAA compared with AD‐like subjects (p = 0.007–0.03). Besides Aβ1‐42, none of the Aβ peptides were decreased in AD‐like subjects compared with controls. All Aβ peptides combined differentiated CAA from AD‐like subjects better (area under the curve [AUC] 0.84) than individual peptide levels (AUC 0.51–0.75). Without Aβ1‐42 in the model (since decreased Aβ1‐42 served as AD‐like selection criterion), the AUC was 0.78 for distinguishing CAA from AD‐like subjects. CAA patients and AD‐like subjects showed distinct disease‐specific CSF Aβ profiles. Peptides shorter than Aβ1‐42 were decreased in CAA patients, but not AD‐like subjects, which could suggest different pathological mechanisms between vascular and parenchymal Aβ accumulation. This study supports the potential use of this panel of CSF Aβ peptides to indicate presence of CAA pathology with high accuracy. [ABSTRACT FROM AUTHOR]

Background: Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer's disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis. Method: Using liquid chromatography coupled to mass-spectrometry (LC–MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset. Results: The correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort. Conclusion: We have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD. [ABSTRACT FROM AUTHOR]

The core cerebrospinal fluid (CSF) biomarkers; total tau (tTau), phospho-tau (pTau), amyloid β 1-42 (Aβ 1-42), and the Aβ 1-42/Aβ 1-40 ratio have transformed Alzheimer's disease (AD) research and are today increasingly used in clinical routine laboratories as diagnostic tools. Fully automated immunoassay instruments with ready-to-use assay kits and calibrators has simplified their analysis and improved reproducibility of measurements. We evaluated the analytical performance of the fully automated immunoassay instrument LUMIPULSE G (Fujirebio) for measurement of the four core AD CSF biomarkers and determined cutpoints for AD diagnosis. Comparison of the LUMIPULSE G assays was performed with the established INNOTEST ELISAs (Fujirebio) for hTau Ag, pTau 181, β-amyloid 1-42, and with V-PLEX Plus Aβ Peptide Panel 1 (6E10) (Meso Scale Discovery) for Aβ 1-42/Aβ 1-40, as well as with a LC-MS reference method for Aβ 1-42. Intra- and inter-laboratory reproducibility was evaluated for all assays. Clinical cutpoints for Aβ 1-42, tTau, and pTau was determined by analysis of three cohorts of clinically diagnosed patients, comprising 651 CSF samples. For the Aβ 1-42/Aβ 1-40 ratio, the cutpoint was determined by mixture model analysis of 2,782 CSF samples. The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The repeatability (intra-laboratory) CVs ranged between 2.0 and 5.6%, with the highest variation observed for β-amyloid 1-40. The reproducibility (inter-laboratory) CVs ranged between 2.1 and 6.5%, with the highest variation observed for β-amyloid 1-42. The clinical cutpoints for AD were determined to be 409 ng/L for total tau, 50.2 ng/L for pTau 181, 526 ng/L for β-amyloid 1-42, and 0.072 for the Aβ 1-42/Aβ 1-40 ratio. Our results suggest that the LUMIPULSE G assays for the CSF AD biomarkers are fit for purpose in clinical laboratory practice. Further, they corroborate earlier presented reference limits for the biomarkers. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is biologically heterogeneous, and detailed understanding of the processes involved in patients is critical for development of treatments. CSF contains hundreds of proteins, with concentrations reflecting ongoing (patho)physiological processes. This provides the opportunity to study many biological processes at the same time in patients. We studied whether Alzheimer's disease biological subtypes can be detected in CSF proteomics using the dual clustering technique non-negative matrix factorization. In two independent cohorts (EMIF-AD MBD and ADNI) we found that 705 (77% of 911 tested) proteins differed between Alzheimer's disease (defined as having abnormal amyloid, n = 425) and controls (defined as having normal CSF amyloid and tau and normal cognition, n = 127). Using these proteins for data-driven clustering, we identified three robust pathophysiological Alzheimer's disease subtypes within each cohort showing (i) hyperplasticity and increased BACE1 levels; (ii) innate immune activation; and (iii) blood-brain barrier dysfunction with low BACE1 levels. In both cohorts, the majority of individuals were labelled as having subtype 1 (80, 36% in EMIF-AD MBD; 117, 59% in ADNI), 71 (32%) in EMIF-AD MBD and 41 (21%) in ADNI were labelled as subtype 2, and 72 (32%) in EMIF-AD MBD and 39 (20%) individuals in ADNI were labelled as subtype 3. Genetic analyses showed that all subtypes had an excess of genetic risk for Alzheimer's disease (all P > 0.01). Additional pathological comparisons that were available for a subset in ADNI suggested that subtypes showed similar severity of Alzheimer's disease pathology, and did not differ in the frequencies of co-pathologies, providing further support that found subtypes truly reflect Alzheimer's disease heterogeneity. Compared to controls, all non-demented Alzheimer's disease individuals had increased risk of showing clinical progression (all P < 0.01). Compared to subtype 1, subtype 2 showed faster clinical progression after correcting for age, sex, level of education and tau levels (hazard ratio = 2.5; 95% confidence interval = 1.2, 5.1; P = 0.01), and subtype 3 at trend level (hazard ratio = 2.1; 95% confidence interval = 1.0, 4.4; P = 0.06). Together, these results demonstrate the value of CSF proteomics in studying the biological heterogeneity in Alzheimer's disease patients, and suggest that subtypes may require tailored therapy. [ABSTRACT FROM AUTHOR]

Aberrant tau phosphorylation is a hallmark in Alzheimer's disease (AD), believed to promote formation of paired helical filaments, the main constituent of neurofibrillary tangles in the brain. While cerebrospinal fluid (CSF) levels of total tau and tau phosphorylated at threonine residue 181 (pThr181) are established core biomarkers for AD, the value of alternative phosphorylation sites, which may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardized detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF, we have applied an innovative mass spectrometry (MS) workflow, TMTcalibratortrademark, to enrich and enhance the detection of phosphoproteome components of AD brain tissue in CSF, and enable the quantitation of these analytes. We aimed to identify which tau species present in the AD brain are also detectable in CSF and which, if any, are differentially regulated with disease. Over 75% coverage of full-length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopeptides were upregulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Use of the TMTcalibratortrademark workflow dramatically improved our ability to detect tau-derived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD is warranted and is currently being undertaken. [ABSTRACT FROM AUTHOR]

Describes a novel matrix-assisted laser desorption/ionization (MALDI) sample preparation technique for peptide analysis using the matrix alpha-cyano-4-hydroxycinnamic acid. Protocol for MALDI mass spectrometry peptide analysis in proteomics; Alpha-cyano-4-hydroxycinnamic acid affinity sample preparation.

Abstract: We have used a non-contact piezo-electric liquid dispensing workstation to transfer 200pL aliquots of both a sample solution containing 12 peptides, each at a concentration of 5 or 50fmol/μL, and dilution buffer onto microcrystalline spots of α-cyano-4-hydroxycinnamic acid for thin-layer preparations on prestructured MALDI sample supports. This approach allowed us to systematically investigate the dependence of detection sensitivity on sample volume, spot size, concentration, as well as sample losses. We demonstrate that 1amol of the peptide mixture was sufficient to detect all components if it was prepared in a volume of 0.2nL across a spot of ca. 180μm diameter. If the sample was diluted on the target to 200nL and prepared on a spot of 400μm diameter, 10amol could be detected and if the sample volume was increased to 1μL, 50amol were required to detect all components. If the peptide mixture instead was diluted in a sample vial and 1μL of that solution was prepared on a spot of 400μm diameter, 50amol of the peptide mixture could be detected as well and the signal-to-noise ratios of the analyte signals were comparable to those observed for the on-target dilution experiment. [Copyright &y& Elsevier]

We used MALDI-MS to study the interaction of amyloid β (Aβ) peptides with alpha-2-macroglobulin (α2M). The binding of amyloid beta (Aβ) peptides to alpha-2-macroglobulin (α2M) was found to inhibit the ability of trypsin to cleave out the peptide α2M 705–715 (Pep-α2M) from α2M. This was observed with both purified α2M and α2M in human serum. We found that Aβ 1–38, Aβ1-40, and Aβ 1–42, all inhibit the interaction of α2M with trypsin, with inhibition rate independent of the length of the Aβ peptide. Further, we show that for complete inhibition, two peptide molecules must be attached to one α2M molecule; one for each of its two subunits. A region was revealed within the Aβ sequence, in which proteolytic cleavage (Lys-28) and oxidation (Met-35) lead to a loss of their ability to inhibit the interaction of trypsin with α2M. Furthermore, we show that after the formation of a trypsin complex with α2M and cleavage of α2M to produce the α2M 705–715, Aβ peptides continue to bind to the protein in the same proportions. However, Aβ peptides treated with DMSO lost their ability to bind to α2M and thereby to inhibit the interaction of trypsin with α2M. While maintaining their primary structure, such an effect can be explained only by conformational changes in the peptides, suggesting the possibility to use our analytical approach to distinguish between conformational isomers of Aβ peptides. [Display omitted] • Amyloid beta peptides inhibit the interaction of α-2-macroglobulin with trypsin. • The inhibition can be measured directly by MALDI-MS. • Two Aβ peptide molecules bind per α2M molecule. • The interaction site appears to be located in the C-terminal region of Aβ • Binding to α2M is dependent on Aβ conformation. Summary Paragraph : We present a method for studying the interaction of amyloid β (Aβ) peptides with α-2-macroglobulin (α2M) by MALDI-MS. [ABSTRACT FROM AUTHOR]

Highlights: [•] A mass spectrometry method for detection of Aβ peptides in plasma was developed. [•] Sixteen truncated Aβ peptides were reproducibly detected in plasma. [•] The method has the potential to be used as a read out in clinical trials. [Copyright &y& Elsevier]