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Clinical Chemistry 51: 973-980, 2005. First published April 21, 2005; 10.1373/clinchem.2004.047308
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2005;51:973-980.)
© 2005 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Sven Baumann2, Uta Ceglarek2, Georg Martin Fiedler, Jan Lembcke, Alexander Leichtle and Joachim Thierya

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany.

aAddress correspondence to this author at: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany. Fax 49-341-9722209; e-mail thiery{at}medizin.uni-leipzig.de.

Background: Magnetic bead purification for the analysis of low-abundance proteins in body fluids facilitates the identification of potential new biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aims of our study were to establish a proteome fractionation technique and to validate a standardized blood sampling, processing, and storage procedure for proteomic pattern analysis.

Methods: We used magnetic bead separation for proteome profiling of human blood by MALDI-TOF MS (mass range, 1000–10 000 Da) and studied the effects on the quality and reproducibility of the proteome analysis of anticoagulants, blood clotting, time and temperature of sample storage, and the number of freeze–thaw cycles of samples.

Results: The proteome pattern of human serum was characterized by ~350 signals in the mass range of 1000–10 000 Da. The proteome profile showed time-dependent dynamic changes before and after centrifugation of the blood samples. Serum mass patterns differed between native samples and samples frozen once. The best reproducibility of proteomic patterns was with a single thawing of frozen serum samples.

Conclusion: Application of the standardized preanalytical blood sampling and storage procedure in combination with magnetic bead-based fractionation decreases variability of proteome patterns in human serum assessed by MALDI-TOF MS.




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