Characterization of the Low Molecular Weight Human Serum Proteome

doi:10.1074/mcp.M300031-MCP200

Characterization of the Low Molecular Weight Human Serum Proteome*S

From the SAIC-Frederick Inc., Laboratory of Proteomics and Analytical Technologies, Mass Spectrometry Center, National Cancer Institute at Frederick, Frederick, MD 21702-1201

‡To whom correspondence should be addressed: Biomedical Proteomics Program, SAIC-Frederick Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702-1201. Tel.: 301-846-7286; Fax: 301-846-6037; E-mail: veenstrancifcrf.gov

Abstract

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.

Footnotes

Published, MCP Papers in Press, August 13, 2003, DOI 10.1074/mcp.M300031-MCP200
↵*S This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400. 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.
↵1 The abbreviations used are: LMW, low molecular weight; μLC, micro-capillary reverse-phase liquid chromatography; SELDI, surface-enhanced laser/desorption ionization; TOF, time-of-flight; MS, mass spectrometry; MWCO, molecular weight cutoff; MS/MS, tandem mass spectrometry; IT-MS, ion-trap mass spectrometer; CID, collision-induced dissociation; SCX, strong cation exchange.
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The on-line version of this article (available at http://www.mcponline.org ) contains supplemental data.
- Received April 7, 2003.
- Revision received August 12, 2003.