Summary Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. We describe quantitative mass spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers of which 77 provided high-quality data. Integrated analyses allowed insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. The 5q trans effects were interrogated against the Library of Integrated Network-based Cellular Signatures, thereby connecting CETN3 and SKP1 loss to elevated expression of EGFR, and SKP1 loss also to increased SRC. Global proteomic data confirmed a stromal-enriched group in addition to basal and luminal clusters and pathway analysis of the phosphoproteome identified a G Protein-coupled receptor cluster that was not readily identified at the mRNA level. Besides ERBB2, other amplicon-associated, highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Summary We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology.

SUMMARY To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSC). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease such as how different copy number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, as well as the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.Proteomic technologies based on mass spectrometry (MS) have emerged as preferred components of a strategy for discovery of diagnostic, prognostic and therapeutic protein biomarkers. Because of the stochastic sampling of proteomes in unbiased analyses and the associated high false-discovery rate, tens to hundreds of potential biomarkers are often reported in discovery studies. Those few that will ultimately show sufficient sensitivity and specificity for a given medical condition must thus be culled from lengthy lists of candidates -a particularly challenging aspect of the biomarker-development pipeline and currently its main limiting step. In this context, it is highly desirable to verify, by more targeted quantitative methods, the levels of candidate biomarkers in body fluids, cells, tissues or organs from healthy individuals and affected patients in large enough sample numbers to confirm statistically relevant differences 1, 2. Verification of novel biomarkers has relied primarily on the use of sensitive, specific, high-throughput immunoassays, whose development depends critically on the availability of suitable well-characterized antibodies. However, antibody reagents of sufficient specificity and sensitivity to assay novel protein biomarkers in plasma are generally not available. The high cost and long development time required to generate high-quality immunoassay reagents, as well as technical limitations in multiplexing immunoassays for panels of biomarkers, is strong motivation to develop more straightforward quantitative approaches exploiting the sensitivity and molecular specificity of mass spectrometry.Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution (SID)-MS for direct quantification of proteins in cell lysates as well as human plasma and serum has been shown to have considerable promise 3- RESULTS Study de...

Highlights d Systematic identification of colon cancer-associated proteins and phosphosites d Proteomics-supported neoantigens and cancer/testis antigens in 78% of the tumors d Rb phosphorylation is an oncogenic driver and a putative target in colon cancer d Glycolysis inhibition may render MSI tumors more sensitive to checkpoint blockade

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 From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts; §Eli

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and

Highlights d Comprehensive LUAD proteogenomics exposes multi-omic clusters and immune subtypes d Phosphoproteomics identifies candidate ALK-fusion diagnostic markers and targets d Candidate drug targets: PTPN11 (EGFR), SOS1 (KRAS), neutrophil degranulation (STK11) d Phospho and acetyl modifications denote tumor-specific markers and druggable proteins