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.

Parallel reaction monitoring using quadrupole‐Orbitrap mass spectrometer: Principle and applications

“…In a PRM event, all ions resulting from the fragmentation of a single, or several, precursor ions are measured simultaneously in one MS/MS scan. The technique represents a true alternative to SRM for quantitative analyses, especially in complex samples such as bodily fluids [20,22], and its application in the field of proteomics has progressively gained attention [21][22][23][24][25][26].…”

Chemical Glycoproteomics

“…345 This effort has generated a number of public resources for targeted proteomics, including Skyline, the SRMAtlas, and the CPTAC Assay portal. 348–350 Recently, targeted proteomics was extended to glycoproteins through MRM-based quantitation of glycoforms, 351 PRM assays for N-glycosites, 352 and the introduction of an N-glycosite SRMAt-las. 353 The SRMAtlas was critical in the analysis of human breast tumor samples to identify potential N-glycoprotein biomarkers.…”

Advances in targeted proteomics and applications to biomedical research

“…The combination of immunoaffinity purification and PRM (i.e., immuno-PRM) allows fast screening driver mutations in tissue and tumor markers in human plasma [144]. Very recently, Thomas et al developed multiplexed PRM assays for quantification of 43 N-linked glycositecontaining peptides in prostate patient sera [86]. A total 41 N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified with four proteins showing differential significance between nonaggressive and aggressive prostate cancer patient sera.…”