title: Unravelling in vitro variables of major importance for the outcome of mass spectrometry-based serum proteomics.
abstract: The use of mass spectrometry (MS) for analysing low-molecular weight proteins and peptides from biological fluids has a great, yet not fully realized, potential for biomarker discovery. To prune MS-data as much as possible for non-relevant non-biological variation the development of standardized protocols for handling and processing the samples before MS and adjusting data after MS to compensate for method-induced variability are warranted. This calls for knowledge about how different variables contribute to MS-based proteome analyses. In addition, identification of the peptides involved in pre-analytical variation will be helpful in evaluating the clinical significance of predictive models derived from MS data. Using human sera, extraction by weak cation-exchange magnetic beads, and analysis by MALDI-TOF MS we here evaluated pre-analytical variation and identify peptides involved in this. The influences of humidity, temperature, and time for preparation of sera on spectral changes were evaluated. Also, the reproducibility of the methods and the effect of a baseline correction procedure were examined. Low temperatures, short handling times, and a baseline correction procedure minimize the contribution of artifacts to sample variability as observed by MS. The complement split product C3f and fragments thereof appear to be sensitive indicators of sample handling induced modifications. Other peptides that are indicative of such variability are fibrin and kininogen fragments. Using strict experimental guidelines as well as standardized sample collection procedures it is possible to obtain reproducible peak intensities and positions in serum mass profiling using magnetic bead-based fractionation and MALDI-TOF MS.
authors.affiliation: Department of Autoimmunology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark. mwn@ssi.dk
authors.forename: Mikkel
authors.initials: M
authors.lastname: West-Nørager
authors.forename: Christian Dahl
authors.initials: CD
authors.lastname: Kelstrup
authors.forename: Christian
authors.initials: C
authors.lastname: Schou
authors.forename: Estrid V
authors.initials: EV
authors.lastname: Høgdall
authors.forename: Claus K
authors.initials: CK
authors.lastname: Høgdall
authors.forename: Niels H H
authors.initials: NH
authors.lastname: Heegaard
mesh_terms: D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000302:isolation & purification*; D001800:Blood Specimen Collection; D002853:Chromatography, Liquid; D019295:Computational Biology; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D010449:Peptide Mapping; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015415:Biomarkers; D001798:Blood Proteins