A pilot study to evaluate the application of a generic protein standard panel for quality control of biomarker detection technologies

<p>Abstract</p> <p>Background</p> <p>Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used.</p> <p>Findings</p> <p>In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL) of varying abundance was prepared as a quality control (QC) material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery^®(MSD^®) platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability.</p> <p>Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers.</p> <p>Conclusions</p> <p>We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.</p

Analysis of Overlapped and Noisy Hydrogen/Deuterium Exchange Mass Spectra

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1007/s13361-013-0727-5.Noisy and overlapped mass spectrometry data hinders the sequence coverage that can be obtained from Hydrogen Deuterium exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are reencountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining exchange data for 42 additional peptides, improving the sequence coverage by 11%. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2

The Path to Clinical Proteomics Research: Integration of Proteomics, Genomics, Clinical Laboratory and Regulatory Science

Better biomarkers are urgently needed to cancer detection, diagnosis, and prognosis. While the genomics community is making significant advances in understanding the molecular basis of disease, proteomics will delineate the functional units of a cell, proteins and their intricate interaction network and signaling pathways for the underlying disease. Great progress has been made to characterize thousands of proteins qualitatively and quantitatively in complex biological systems by utilizing multi-dimensional sample fractionation strategies, mass spectrometry and protein microarrays. Comparative/quantitative analysis of high-quality clinical biospecimen (e.g., tissue and biofluids) of human cancer proteome landscape has the potential to reveal protein/peptide biomarkers responsible for this disease by means of their altered levels of expression, post-translational modifications as well as different forms of protein variants. Despite technological advances in proteomics, major hurdles still exist in every step of the biomarker development pipeline. The National Cancer Institute's Clinical Proteomic Technologies for Cancer initiative (NCI-CPTC) has taken a critical step to close the gap between biomarker discovery and qualification by introducing a pre-clinical "verification" stage in the pipeline, partnering with clinical laboratory organizations to develop and implement common standards, and developing regulatory science documents with the US Food and Drug Administration to educate the proteomics community on analytical evaluation requirements for multiplex assays in order to ensure the safety and effectiveness of these tests for their intended use

Two-dimensional SDS-PAGE fractionation of biological samples for biomarker discovery

Two-dimensional electrophoresis is still a very valuable tool in proteomics, due to its reproducibility and its ability to analyze complete proteins. However, due to its sensitivity to dynamic range issues, its most suitable use in the frame of biomarker discovery is not on very complex fluids such as plasma, but rather on more proximal, simpler fluids such as CSF, urine, or secretome samples. Here, we describe the complete workflow for the analysis of such dilute samples by two-dimensional electrophoresis, starting from sample concentration, then the two-dimensional electrophoresis step per se, ending with the protein detection by fluorescence

ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use

HDL Proteome in Hemodialysis Patients: A Quantitative Nanoflow Liquid Chromatography-Tandem Mass Spectrometry Approach

Aside from a decrease in the high-density lipoprotein (HDL) cholesterol levels, qualitative abnormalities of HDL can contribute to an increase in cardiovascular (CV) risk in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Dysfunctional HDL leads to an alteration of reverse cholesterol transport and the antioxidant and anti-inflammatory properties of HDL. In this study, a quantitative proteomics approach, based on iTRAQ labeling and nanoflow liquid chromatography mass spectrometry analysis, was used to generate detailed data on HDL-associated proteins. The HDL composition was compared between seven chronic HD patients and a pool of seven healthy controls. To confirm the proteomics results, specific biochemical assays were then performed in triplicate in the 14 samples as well as 46 sex-matched independent chronic HD patients and healthy volunteers. Of the 122 proteins identified in the HDL fraction, 40 were differentially expressed between the healthy volunteers and the HD patients. These proteins are involved in many HDL functions, including lipid metabolism, the acute inflammatory response, complement activation, the regulation of lipoprotein oxidation, and metal cation homeostasis. Among the identified proteins, apolipoprotein C-II and apolipoprotein C-III were significantly increased in the HDL fraction of HD patients whereas serotransferrin was decreased. In this study, we identified new markers of potential relevance to the pathways linked to HDL dysfunction in HD. Proteomic analysis of the HDL fraction provides an efficient method to identify new and uncharacterized candidate biomarkers of CV risk in HD patients

Genome-Wide Association Study Confirming Association of HLA-DP with Protection against Chronic Hepatitis B and Viral Clearance in Japanese and Korean

Hepatitis B virus (HBV) infection can lead to serious liver diseases, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC); however, about 85–90% of infected individuals become inactive carriers with sustained biochemical remission and very low risk of LC or HCC. To identify host genetic factors contributing to HBV clearance, we conducted genome-wide association studies (GWAS) and replication analysis using samples from HBV carriers and spontaneously HBV-resolved Japanese and Korean individuals. Association analysis in the Japanese and Korean data identified the HLA-DPA1 and HLA-DPB1 genes with Pmeta = 1.89×10−12 for rs3077 and Pmeta = 9.69×10−10 for rs9277542. We also found that the HLA-DPA1 and HLA-DPB1 genes were significantly associated with protective effects against chronic hepatitis B (CHB) in Japanese, Korean and other Asian populations, including Chinese and Thai individuals (Pmeta = 4.40×10−19 for rs3077 and Pmeta = 1.28×10−15 for rs9277542). These results suggest that the associations between the HLA-DP locus and the protective effects against persistent HBV infection and with clearance of HBV were replicated widely in East Asian populations; however, there are no reports of GWAS in Caucasian or African populations. Based on the GWAS in this study, there were no significant SNPs associated with HCC development. To clarify the pathogenesis of CHB and the mechanisms of HBV clearance, further studies are necessary, including functional analyses of the HLA-DP molecule