PROJECT SUMMARY
The overall objective of the PNNL Proteome Characterization Center (PCC) is to comprehensively characterize
human tumor samples provided by the National Cancer Institute (NCI), and to integrate the multi-omic
measurements to support improved understanding of the molecular changes that characterize cancer, and do
so in the context of clinical outcome. PNNL has participated in the NCI’s Clinical Proteomic Tumor Analysis
Consortium (CPTAC) as a PCC for the past ten years, with responsibility for comprehensive proteogenomic
characterization of high-grade serous ovarian, colon, and endometrial cancers, and glioblastoma. The planned
PNNL PCC will build on those achievements to extend and advance the CPTAC mission of comprehensive
proteogenomic characterization of human cancers to additional cancer types, meeting or exceeding CPTAC
key expectations or requirements for sample throughput, coverage, sample size, and data quality. Utilizing an
advanced analytical platform, PNNL plans to add analysis of both acetylome and ubiquitinome to the
phosphoproteome of prospectively collected human tumors, to betters illuminate key biochemical processes
related to protein-protein interactions, protein degradation, and signal transduction. We will also complement
the core proteome and post-translational modification (PTM)-ome analysis with global metabolome and
lipidome analysis, as well as selected data driven spatial or single-cell proteomics analysis. This will provide
additional critical insights on potential metabolic vulnerabilities and tumor heterogeneity as well as
microenvironment contributions. This multi-omic analysis strategy will also be applied to preclinical samples,
such as cell lines, organoids and patient-derived xenografts. We will also develop targeted mass spectrometric
assays using input from the CPTAC consortium, and particularly the Proteogenomic Data Analysis Centers
(PGDACs), to prioritize targets for further exploring important mechanistic proteomic changes in independent
cohort(s). Throughout this work our measurements will benefit from further performance increases (e.g.,
sensitivity and throughput) based on refining, validating and implementing developments from both PNNL and
the other CPTAC Centers.
The PNNL PCC will identify promising cancer signatures and signaling networks through proteomic and
metabolomic analysis of human biospecimens and relevant preclinical samples for 2-3 cancer types selected
by the CPTAC, using state-of-the-art liquid chromatography-tandem mass spectrometry instrumentation, highly
multiplexed isobaric mass-tag labeling (TMT 16-plex), and integrated sample workflows, as well as additional
advanced metabolomic, spatial and single-cell proteomic planforms, at a throughput of 300 samples per year.
We will also explore mechanistically important proteomic changes in human specimens and model systems
using cutting-edge targeted proteomic platforms, analytically validated and highly multiplexed targeted assays,
and workflows meeting the CPTAC Tier 2 assay guidelines. Two hundred highly specific, multiplexed targeted
proteomics assays will be developed and used for measurements in 300 samples each year. The PNNL PCC
will accomplish both unbiased and targeted multi-omic characterization of cancers in conjunction with
improving the depth, throughput and quality of both unbiased and targeted data generated by implementing
and deploying relevant new technologies, such as nanoscale PTM, metabolomic analysis, and single-cell
proteomics analysis.
The PNNL PCC will work closely with the other PCCs, PGDACs and PTRCs in the CPTAC network on data
integration and bioinformatics analysis, as well as translational applications.