Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry

Robert G. Harper, Sarah R. Workman, Shayne Schuetzner, Aaron T. Timperman, Jennifer N. Sutton

Research output: Contribution to journalArticlepeer-review

Abstract

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.

Original languageEnglish (US)
Pages (from-to)1299-1306
Number of pages8
JournalElectrophoresis
Volume25
Issue number9
DOIs
StatePublished - May 1 2004
Externally publishedYes

Keywords

  • Isoelectric focusing
  • Mass spectrometry
  • Ultrafiltration

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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