Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux.

BACKGROUND: The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation - altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure. METHODS: The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure. RESULTS: The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions. CONCLUSIONS: The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM).

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi-automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.

Hybrid data acquisition and processing strategies with increased throughput and selectivity: pSMART analysis for global qualitative and quantitative analysis.

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis. The performance of pSMART is compared to published DIA strategies in an experiment that allows the objective assessment of DIA performance with respect to interrogation of previously acquired MS data. The results of this experiment demonstrate that pSMART creates fewer decoy hits than a standard DIA strategy. Moreover, we show that pSMART is more selective, sensitive, and reproducible than either standard DIA or DDA strategies alone.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells.

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.

Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow.

Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.

Model-based discovery of circulating biomarkers.

Proteomic-based biomarker discovery approaches broadly attempt to identify proteins whose basal abundance, or change in abundance in response to a perturbation (e.g., a therapeutic intervention) is able to discriminate between populations of patients. Up until recently, the majority of approaches for discovering circulating biomarkers have focused on directly profiling serum or plasma to identify such proteins. However, the complexity and dynamic range of protein abundance in serum and plasma create a significant challenge for proteomics methods. To overcome these barriers, diverse approaches to simplify or to fractionate serum and plasma have been developed. For some diseases, such as those related to specific organs, there may be useful marker proteins that originate in the organ. Here, we describe an approach for marker discovery that focuses on the profiling of either primary tissue or cell culture models thereof.