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 seven 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 subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intraand interlaboratory 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, participatFrom the A Broad Institute of MIT and Harvard,

BackgroundFollicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.ResultsA total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.ConclusionsThis database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.

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 this review we address the relationship between cytochromes P450 (P450) and HO. This association can affect biology in three distinct ways. First, P450s produce HO as a byproduct either during catalysis or when no substrate is present. This reaction, known as uncoupling, releases reactive oxygen species that may have implications in disease. Second, HO is used as an oxygen-donating co-substrate in peroxygenase and peroxidase reactions catalyzed by P450s. This activity has proven to be important mainly in reactions involving prokaryotic P450s, and investigators have harnessed this reaction with the aim of adaptation for industrial use. Third, HO-dependent inhibition of human P450s has been studied in our laboratory, demonstrating heme destruction and also the inactivating oxidation of the heme-thiolate ligand to a sulfenic acid (-SOH). This reversible oxidative modification of P450s may have implications in the prevention of uncoupling and may give new insights into the oxidative regulation of these enzymes. Research has elucidated many of the chemical mechanisms involved in the relationship between P450 and HO, but the application to biology is difficult to evaluate. Further studies are needed reveal both the harmful and protective natures of reactive oxygen species in an organismal context.