title: Multiplexed Targeted Mass Spectrometry-Based Assays for the Quantification of N-Linked Glycosite-Containing Peptides in Serum.
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 to detect and quantify glycoproteins. Here we describe the development of multiplexed targeted MS assays to quantify N-linked glycosite-containing peptides in serum using parallel reaction monitoring (PRM). Each assay was characterized by its performance metrics and criteria established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. An in-house developed software program, MRMPlus, was used to compute assay performance parameters including specificity, precision, and repeatability. We show that 43 selected N-linked glycosite-containing peptides identified in prostate cancer tissue studies carried out in our group were detected in the sera of prostate cancer patients within the quantitative range of the developed PRM assays. A total of 41 of these formerly N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified based on their relative peak area ratios in human serum during PRM assay development, with 4 proteins showing differential significance in serum from nonaggressive (NAG) vs aggressive (AG) prostate cancer patient serum (n = 50, NAG vs AG). The data demonstrate that the assays can be used for the high throughput and reproducible quantification of a panel of formerly N-linked glycosite-containing peptides. The developed assays can also be used for the quantification of formerly N-linked glycosite-containing peptides in human serum irrespective of disease state.
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Stefani N
authors.initials: SN
authors.lastname: Thomas
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Harlan
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Aiyetan
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Yansheng
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Lori J
authors.initials: LJ
authors.lastname: Sokoll
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich , 8093 Zurich, Switzerland.|Faculty of Science, University of Zurich , 8057 Zurich, Switzerland.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Chan
authors.affiliation: Department of Pathology, Clinical Chemistry Division, Johns Hopkins University School of Medicine , 1550 Orleans Street, CRBII Room 3M03, Baltimore, Maryland 21231, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000368:Aged; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D010455:Peptides / Q000097:blood* / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000097:blood
chemical_list: D010455:Peptides