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Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies.

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

On the basis of discussions with representatives from all sectors of the cancer research community, the National Cancer Institute (NCI) recognizes the immense opportunities to apply proteomics technologies to further cancer research. Validated and well characterized affinity capture reagents (e.g. antibodies, aptamers, and affibodies) will play a key role in proteomics research platforms for the prevention, early detection, treatment, and monitoring of cancer. To discuss ways to develop new resources and optimize current opportunities in this area, the NCI convened the "Proteomic Technologies Reagents Resource Workshop" in Chicago, IL on December 12-13, 2005. The workshop brought together leading scientists in proteomics research to discuss model systems for evaluating and delivering resources for reagents to support MS and affinity capture platforms. Speakers discussed issues and identified action items related to an overall vision for and proposed models for a shared proteomics reagents resource, applications of affinity capture methods in cancer research, quality control and validation of affinity capture reagents, considerations for target selection, and construction of a reagents database. The meeting also featured presentations and discussion from leading private sector investigators on state-of-the-art technologies and capabilities to meet the user community's needs. This workshop was developed as a component of the NCI's Clinical Proteomics Technologies Initiative for Cancer, a coordinated initiative that includes the establishment of reagent resources for the scientific community. This workshop report explores various approaches to develop a framework that will most effectively fulfill the needs of the NCI and the cancer research community.

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 μl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and

Peptide immunoaffinity enrichment coupled to selected reaction monitoring (SRM) mass spectrometry (immuno-SRM) has emerged as a technology with great potential for quantitative proteomic assays. One advantage over traditional immunoassays is the tremendous potential for concurrent quantification of multiple analytes from a given sample (i.e. multiplex analysis). We sought to explore the capacity of the immuno-SRM technique for analyzing large numbers of analytes by evaluating the multiplex capabilities and demonstrating the sequential analysis of groups of peptides from a single sample. To evaluate multiplex analysis, immuno-SRM assays were arranged in groups of 10, 20, 30, 40, and 50 peptides using a common set of reagents. The multiplex immuno-SRM assays were used to measure synthetic peptides added to plasma covering several orders of magnitude concentration. Measurements made in large multiplex groups were highly correlated (r(2) ≥ 0.98) and featured good agreement (bias ≤ 1%) compared with single-plex assays or a 10-plex configuration. The ability to sequentially enrich sets of analyte peptides was demonstrated by enriching groups of 10 peptides from a plasma sample in a sequential fashion. The data show good agreement (bias ≤ 1.5%) and similar reproducibility regardless of enrichment order. These significant advancements demonstrate the utility of immuno-SRM for analyzing large numbers of analytes, such as in large biomarker verification experiments or in pathway-based targeted analysis.