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Title: Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

Abstract

The enormous complexity, wide dynamic range of relative protein abundance of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenges the capabilities of existing analytical methodologies. We describe here the comprehensive analysis of human plasma N-glycoproteins using the combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin, enzymatically digested, and the bound, N-linked glycopeptides were released using peptide-N-glycosidase F. Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A total of 2140 different N-glycopeptides were confidently identified using stringent criteria, covering 371 non-redundant N-glycoproteins with the majority of them being extracellular or membrane proteins. The strategy significantly improved the detection, enabling the identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein ({approx}200 pg/mL), cathepsin L ({approx}1 ng/mL), and transforming growth factor beta 1 ({approx}2 ng/mL). A total of 712 N-glycosylation sites were identifiedmore » and the confidence of these site identifications was further validated by accurate mass measurements using high resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR). This study provides the basis for future high-throughput measurements using the accurate mass and time tag approach.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
876915
Report Number(s):
PNNL-SA-44967
12695; 400412000; TRN: US200608%%335
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of Proteome Research
Additional Journal Information:
Journal Volume: 4; Journal Issue: 6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ABUNDANCE; BLOOD PLASMA; CATHEPSINS; CATIONS; CHEMISTRY; CHROMATOGRAPHY; DETECTION; FRACTIONATION; GLYCOPROTEINS; GROWTH FACTORS; HYDRAZIDES; ION CYCLOTRON-RESONANCE; MASS SPECTROSCOPY; MEMBRANE PROTEINS; MODIFICATIONS; PEPTIDES; PROTEINS; RESOLUTION; Environmental Molecular Sciences Laboratory

Citation Formats

Liu, Tao, Qian, Weijun, Gritsenko, Marina A, Camp, David G, Monroe, Matthew E, Moore, Ronald J, and Smith, Richard D. Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry. United States: N. p., 2005. Web. doi:10.1021/pr0502065.
Liu, Tao, Qian, Weijun, Gritsenko, Marina A, Camp, David G, Monroe, Matthew E, Moore, Ronald J, & Smith, Richard D. Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry. United States. https://doi.org/10.1021/pr0502065
Liu, Tao, Qian, Weijun, Gritsenko, Marina A, Camp, David G, Monroe, Matthew E, Moore, Ronald J, and Smith, Richard D. 2005. "Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry". United States. https://doi.org/10.1021/pr0502065.
@article{osti_876915,
title = {Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry},
author = {Liu, Tao and Qian, Weijun and Gritsenko, Marina A and Camp, David G and Monroe, Matthew E and Moore, Ronald J and Smith, Richard D},
abstractNote = {The enormous complexity, wide dynamic range of relative protein abundance of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenges the capabilities of existing analytical methodologies. We describe here the comprehensive analysis of human plasma N-glycoproteins using the combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin, enzymatically digested, and the bound, N-linked glycopeptides were released using peptide-N-glycosidase F. Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A total of 2140 different N-glycopeptides were confidently identified using stringent criteria, covering 371 non-redundant N-glycoproteins with the majority of them being extracellular or membrane proteins. The strategy significantly improved the detection, enabling the identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein ({approx}200 pg/mL), cathepsin L ({approx}1 ng/mL), and transforming growth factor beta 1 ({approx}2 ng/mL). A total of 712 N-glycosylation sites were identified and the confidence of these site identifications was further validated by accurate mass measurements using high resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR). This study provides the basis for future high-throughput measurements using the accurate mass and time tag approach.},
doi = {10.1021/pr0502065},
url = {https://www.osti.gov/biblio/876915}, journal = {Journal of Proteome Research},
number = 6,
volume = 4,
place = {United States},
year = {Thu Dec 01 00:00:00 EST 2005},
month = {Thu Dec 01 00:00:00 EST 2005}
}