Standardized approach to proteome profiling of human serum based on magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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 des orption/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, 1000-10000 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 similar to 350 signals in the mass range of 1000-10000 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. (c) 2005 American Association for Clinical Chemistry