Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays to 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

The multiplex IO-1 immuno-MRM assay was applied to a panel of tumor tissue and plasma specimens with two aims: (i) evaluate the utility of the assay for measuring endogenous levels of analytes in clinical specimens and (ii) determine sample requirements for analyte detection in tissue where clinical material may be limited. The tissue panel included 110 frozen and 25 FFPE specimens collected from 12 different tumor sites (brain, breast, colorectal, endometrium, head and neck, kidney, lung (squamous cell carcinoma and adenocarcinoma), ovarian, pancreas, and soft tissue sarcoma). The plasma panel consisted of 45 plasma samples from patients with 3 different tumor types (breast, colon, ovarian). We used 500 µg aliquots of the frozen tissue lysates and 100 µL aliquots of plasma as input for the assay. Each specimen was independently digested, enriched for the IO-1 analytes, and analyzed by LC-MRM.

Samples were supplied by the Clinical Proteomics Tumor Analysis Consortium (CPTAC) and Veteran's Administration as anonymized samples from consenting donors collected under IRB-approved protocols. Please see publication for details.