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Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

ABSTRACT: 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 targeting 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.

SUBMITTER: Whiteaker JR

PROVIDER: S-EPMC8632241 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Whiteaker Jeffrey R JR Lundeen Rachel A RA Zhao Lei L Schoenherr Regine M RM Burian Aura A Huang Dongqing D Voytovich Ulianna U Wang Tao T Kennedy Jacob J JJ Ivey Richard G RG Lin Chenwei C Murillo Oscar D OD Lorentzen Travis D TD Thiagarajan Mathangi M Colantonio Simona S Caceres Tessa W TW Roberts Rhonda R RR Knotts Joseph G JG Reading Joshua J JJ Kaczmarczyk Jan A JA Richardson Christopher W CW Garcia-Buntley Sandra S SS Bocik William W Hewitt Stephen M SM Murray Karen E KE Do Nhan N Brophy Mary M Wilz Stephen W SW Yu Hongbo H Ajjarapu Samuel S Boja Emily E Hiltke Tara T Rodriguez Henry H Paulovich Amanda G AG

Frontiers in immunology 20211111

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 ...[more]

PMID: 34858420

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Project description:BackgroundOvarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera.MethodsTo develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates.ResultsWe describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions.ConclusionsA multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.

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Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Publications

Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

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