ac5b02063_si_003.xlsx (58.71 kB)
Multiplexed Targeted Mass Spectrometry-Based Assays for the Quantification of N‑Linked Glycosite-Containing Peptides in Serum
dataset
posted on 2015-11-03, 00:00 authored by Stefani N. Thomas, Robert Harlan, Jing Chen, Paul Aiyetan, Yansheng Liu, Lori J. Sokoll, Ruedi Aebersold, Daniel
W. Chan, Hui ZhangProtein 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.
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MS assaysNAGpeptidedisease state37 proteinsPRM assay development4 proteinsNCI CPTACprostate cancer tissue studiesreaction monitoringassay performance parametersprostate cancer patient serumpeak area ratiossoftware programPRM assaysprostate cancer patientsprotein modificationsSerumProtein glycosylationAGperformance metrics
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