Large-scale inter-laboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma

In multiple sclerosis, oligoclonal bands connect to peripheral B‐cell responses

OBJECTIVE: To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients. METHODS: Mass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients. RESULTS: We find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood–brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related. INTERPRETATION: Our data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier

In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses.

ObjectiveTo determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients.MethodsMass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients.ResultsWe find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood-brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related.InterpretationOur data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier

In multiple sclerosis, oligoclonal bands connect to peripheral B‐cell responses

ObjectiveTo determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients.MethodsMass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients.ResultsWe find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood-brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related.InterpretationOur data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier

TCA cycle metabolic compromise due to an aberrant S-nitrosoproteome in HIV-associated neurocognitive disorder with methamphetamine use

In the brain, both HIV-1 and methamphetamine (meth) use result in increases in oxidative and nitrosative stress. This redox stress is thought to contribute to the pathogenesis of HIV-associated neurocognitive disorder (HAND) and further worsening cognitive activity in the setting of drug abuse. One consequence of such redox stress is aberrant protein S-nitrosylation, derived from nitric oxide, which may disrupt normal protein activity. Here, we report an improved, mass spectrometry-based technique to assess S-nitrosylated protein in human postmortem brains using selective enrichment of S-nitrosocysteine residues with an organomercury resin. The data show increasing S-nitrosylation of tricarboxylic acid (TCA) enzymes in the setting of HAND and HAND/meth use compared with HIV+ control brains without CNS pathology. The consequence is systematic inhibition of multiple TCA cycle enzymes, resulting in energy collapse that can contribute to the neuronal and synaptic damage observed in HAND and meth use

Alterations in the Salivary Proteome and N‑Glycome of Sjögren’s Syndrome Patients

We used isobaric mass tagging (iTRAQ) and lectin affinity capture mass spectrometry (MS)-based workflows for global analyses of parotid saliva (PS) and whole saliva (WS) samples obtained from patients diagnosed with primary Sjögren's Syndrome (pSS) who were enrolled in the Sjögren's International Collaborative Clinical Alliance (SICCA) as compared with two control groups. The iTRAQ analyses revealed up- and down-regulation of numerous proteins that could be involved in the disease process (e.g., histones) or attempts to mitigate the ensuing damage (e.g., bactericidal/permeability increasing fold containing family (BPIF) members). An immunoblot approach applied to independent sample sets confirmed the pSS associated up-regulation of β2-microglobulin (in PS) and down-regulation of carbonic anhydrase VI (in WS) and BPIFB2 (in PS). Beyond the proteome, we profiled the N-glycosites of pSS and control samples. They were enriched for glycopeptides using lectins Aleuria aurantia and wheat germ agglutinin, which recognize fucose and sialic acid/N-acetyl glucosamine, respectively. MS analyses showed that pSS is associated with increased N-glycosylation of numerous salivary glycoproteins in PS and WS. The observed alterations of the salivary proteome and N-glycome could be used as pSS biomarkers enabling easier and earlier detection of this syndrome while lending potential new insights into the disease process

Mass spectrometry-based analyses showing the effects of secretor and blood group status on salivary N-glycosylation

BackgroundThe carbohydrate portions of salivary glycoproteins play important roles, including mediating bacterial and leukocyte adhesion. Salivary glycosylation is complex. Many of its glycoproteins present ABO and Lewis blood group determinants. An individual's genetic complement and secretor status govern the expression of blood group antigens. We queried the extent to which salivary glycosylation varies according to blood group and secretor status. First, we screened submandibular/sublingual and parotid salivas collected as ductal secretions for reactivity with a panel of 16 lectins. We selected three lectins that reacted with the largest number of glycoproteins and one that recognized uncommon lactosamine-containing structures. Ductal salivas representing a secretor with complex blood group expression and a nonsecretor with a simple pattern were separated by SDS-PAGE. Gel slices were trypsin digested and the glycopeptides were individually separated on each of the four lectins. The bound fractions were de-N-glycosylated. LC-MS/MS identified the original glycosylation sites, the peptide sequences, and the parent proteins.ResultsThe results revealed novel salivary N-glycosites and glycoproteins not previously reported. As compared to the secretor, nonsecretor saliva had higher levels of N-glycosylation albeit with simpler structures.ConclusionsTogether, the results suggested a molecular basis for inter-individual variations in salivary protein glycosylation with functional implications for oral health