High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

doi:10.1074/mcp.M400090-MCP200

High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry *S

From the ^‡Institute for Systems Biology, Seattle, Washington 98103, ^¶Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, ^‖Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, and ^**Swiss Federal Institute of Technology (ETH) Zurich, and Faculty of Natural Sciences, University of Zurich, CH-8093 Zurich, Switzerland

§To whom correspondence should be addressed. E-mail: hzhang{at}systemsbiology.org

Abstract

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

Footnotes

Published, MCP Papers in Press, December 17, 2004, DOI 10.1074/mcp.M400090-MCP200
↵1 In this paper, the term serum is used to indicate serum or plasma.
↵2 The abbreviations used are: SELDI, surface-enhanced laser desorption ionization; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; MALDI, matrix-assisted laser desorption ionization; CID, collision-induced dissociation; TOF, time-of-flight; QTOF, quadrupole time-of-flight; CV, coefficient of variance; DMBA, 7,12-dimethylbenz[a]anthracene; HPLC, high performance liquid chromatography.
↵3 X. J. Li, E. C. Yi, H. Zhang, and R. Aebersold, manuscript in preparation.
↵* This work was supported in part by NCI, National Institutes of Health Grants R33 and CA93302, by Grant N01-HV-28179 from the NHLBI, National Institutes of Health, Proteomics Initiative, and by a sponsored research agreement from Industrial Technology Research Institute in Taiwan. The Institute for Systems Biology was supported by a generous gift from Merck & Co. The LC-FTICR analyses at PNNL were supported by NIH National Center for Research Resources (RR-18522). Pacific Northwest National Laboratory is operated by the Battelle Memorial Institute for the U. S. Department of Energy through Contract DE-AC06-76RLO1830. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
- Received July 12, 2004.
- Revision received November 30, 2004.