Stefani Thomas (Presenter)
Johns Hopkins University
Bio: Stefani Thomas earned her B.A. in Biological Sciences from Dartmouth College and her Ph.D. in Pharmaceutical Sciences with a focus on Biological Mass Spectrometry from the University of Southern California under the mentorship of Austin Yang. Stefani was the Manager of the University of Maryland, Baltimore’s Greenebaum Cancer Center Proteomics Shared Service from 2007 - 2011. In 2012, Stefani joined the laboratory of Robert Cotter in the Department of Pharmacology and Molecular Sciences at Johns Hopkins University. Since July 2013, Stefani has been a Research Associate in the Center for Biomarker Discovery and Translation in the Department of Pathology at Johns Hopkins. Her current research interests include the application of quantitative mass spectrometry-based proteomic strategies to elucidate the biology of ovarian and prostate cancer.
Authorship: Stefani N. Thomas (1), Robert Harlan (1), Jing Chen (1), Paul Aiyetan (1), Yansheng Liu (2), Lori J. Sokoll (1), Ruedi Aebersold (2,3), Daniel W. Chan (1), Hui Zhang (1)
(1) Johns Hopkins University School of Medicine, Baltimore, MD, USA; (2) ETH Zurich, Zurich, Switzerland; (3) University of Zurich, Zurich, Switzerland
| Short Abstract Protein glycosylation is one of the most common protein modifications, and the quantitative analysis of glycoproteins has the potential to reveal biological functions and their association with disease. However, the high throughput accurate quantification of glycoproteins is technically challenging due to the scarcity of robust assays for their detection and quantification. We developed and characterized 43 multiplexed parallel reaction monitoring (PRM) assays that were used to quantify formerly N-linked glycosite-containing peptides from 37 proteins in serum from prostate cancer patients. The assays were characterized by performance metrics and criteria established by the NCI’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. |
Long Abstract
INTRODUCTION
Glycosylation is one of the most common protein modifications, and aberrant glycosylation has been implicated in carcinogenesis.(1-5) The quantification of post-translational modifications (PTMs) in individual proteins is technically challenging, in part due to the scarce availability of specific assays required to confidently detect the respective site of modification. However, targeted mass spectrometry-based assays such as parallel reaction monitoring (PRM) for the site-specific quantification of protein PTMs can be developed with relative ease.
The widespread availability of validated targeted MS-based assays has been recognized as a critical pre-requisite to quantify proteins and to generally increase the reproducibility of data between laboratories and studies. The National Cancer Institute (NCI) has promoted the standardization and analytical validation of targeted MS-based quantification of peptides through the Clinical Proteomic Tumor Analysis Consortium (CPTAC).(6,7) Following the framework for targeted MS-based assay “fit-for-purpose” validation,(8) we developed and characterized 43 “Tier 2” PRM assays targeting N-linked glycosite-containing peptides from serum proteins. Tier 2 targeted MS-based assays are precise, relative quantitative assays with established performance that are most suitable for target verification, wherein relative changes in the levels of large numbers of targeted analytes are precisely and consistently measured across samples for non-clinical purposes.
Prostate cancer carcinogenesis is associated with aberrant glycosylation, and the majority of prostate cancer biomarkers are glycoproteins. The identification of proteins whose relative levels of abundance can differentiate aggressive (AG) from non-aggressive (NAG) prostate cancer is an important step in prostate cancer biomarker development. Our fully characterized PRM assays were deployed for the analysis of serum samples from prostate cancer patients with different disease states to detect differences in the relative abundance of N-linked glycosite-containing peptides.
METHODS
Assay development was conducted using crude stable isotope-labeled (SIS) N-linked glycosite-containing heavy-isotope-labeled peptide standards and endogenous N-linked glycosite-containing peptides enriched from commercially available human serum. The mixture of endogenous and SIS peptides was analyzed by LC-PRM using a Dionex UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). SIS peptides were spiked into and serially diluted with a biological matrix consisting of N-linked glycosite-containing peptides enriched from commercially available human serum using an automated format of the solid-phase extraction of N-linked glycopeptides (SPEG) method.(9,10) Reverse response curves were generated for each peptide to determine the linear range of its corresponding assay. Peak area ratios (heavy/light) were used as the dependent variables to generate the response curves. The assays were characterized based on several metrics including Linearity, Carry-Over, Partial Validation of Specificity, Intra-day Assay CV, Inter-day Assay CV, and Total Assay CV. All Skyline-processed data for the assays that passed the precision criteria of CV ≤20% are available at https://panoramaweb.org/labkey/project/CPTAC%20Assay%20Portal/JHU_DChan_HZhang_ZZhang/Serum_QExactive_GlycopeptideEnrichedPRM/begin.view? Assay details, assay parameters, response curves, repeatability data, detailed standard operating protocols, and additional assay-specific resources can be located on the CPTAC assay portal https://assays.cancer.gov/ using the search term “Johns Hopkins University.”
The serum samples for PRM assay deployment were obtained from the Johns Hopkins Clinical Chemistry laboratory specimen bank. The samples were from three groups of a total of 75 patients: Men who underwent radical prostatectomy (RP) for prostate cancer and had characteristics suggestive of aggressive or non-aggressive disease, and non-cancer/biopsy negative.
RESULTS
The 43 N-linked glycosite-containing peptides selected for PRM assay development were previously identified from prostate cancer tissue discovery-phase proteomic studies conducted by our group.(11,12) Forty-one PRM assays targeting N-linked glycosite-containing peptides had Intra-, Inter- and Total Assay CVs <20%, and they were deployed using serum from males without prostate cancer and males with NAG or AG prostate cancer. The patient-derived serum samples were processed via SPEG in a manner identical to the commercial human serum used for the assay characterization experiments to enrich N-linked glycosite-containing peptides. Prior to PRM analysis, each sample was spiked with a mixture of the 41 heavy-isotope labeled peptide standards to enable peak area comparison. Four N-linked glycosite-containing peptides had significantly higher levels (p<0.05) in the serum from the NAG vs. AG patient groups: AFNSTLPTMAQMEK (CD44 antigen; p=0.040); EEQFNSTFR (Immunoglobulin γ-2 heavy chain; p=0.041); GAFISNFSMTVDGK (Inter-alpha-trypsin inhibitor heavy chain H2, ITIH2; p=0.010); and INNTHALVSLLQNLNK (Cadherin-13; p=0.016). None of the 41 N-linked glycosite-containing peptides had relative abundance levels that were significantly different between the prostate cancer (AG, NAG) and non-cancer (Neg.) groups.
CONCLUSIONS
The PRM assays we developed in this study can be used to evaluate the relative level of N-linked glycosite-containing peptides in human serum regardless of the disease state of the patients from which the serum is obtained. The public availability of the assay data will enable other researchers to evaluate the assay performance prior to deploying the assays in their own laboratories.
References & Acknowledgements:
REFERENCES
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ACKNOWLEDGMENTS
This work was supported in part by the National Institutes of Health under grants and contracts from the National Cancer Institute Clinical Proteomics Tumor Analysis Consortium (CPTAC, U24CA160036 to H.Z. and D.C.) and the Early Detection Research Network (EDRN, U01CA152813 to H.Z. and U24CA115102 to D.C.); the National Heart, Lung and Blood Institute Programs of Excellence in Glycosciences (P01HL107153 to H.Z.) and NHLBI Proteomic Center (N01-HV-00240 to H.Z.); and from the Swiss National Science Foundation (grant# 3100A0-107679 to R.A.).
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