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Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

¹ 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, Liebigstr. 27, D-04103 Leipzig, Germany. Fax 49-341-9722209; e-mail thiery{at}medizin.uni-leipzig.de.

Background: Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).

Methods: MBs with defined surface functionalities (hydrophobic interaction, cation exchange, and metal ion affinity) were used for peptide fractionation of urine. Mass accuracy and imprecision were calculated for 9 characteristic mass signals (M_r, 1000–10 000). Exogenous variables (instrument performance, urine sampling/storage conditions, freezing conditions, and freeze-thaw cycles) and endogenous variables (pH, urine salt and protein concentrations, and blood and bacteria interferences) were investigated with urine samples from 10 male and 10 female volunteers.

Results: We detected 427 different mass signals in the urine of healthy donors. Within- and between-day imprecision in relative signal intensities ranged from 1% to 14% and from 4% to 16%, respectively. Weak cation-exchange and metal ion affinity MB preparations required adjustment of the urinary pH to 7. Storage time, storage temperature, the number of freeze-thaw cycles, and bacterial and blood contamination significantly influenced urine peptide patterns. Individual urine peptide patterns differed significantly within and between days. This imprecision was diminished by normalization to a urinary protein content of 3.5 µg.

Conclusion: This reliable pretreatment protocol allows standardization of preanalytical modalities and facilitates reproducible peptidome profiling of human urine by means of MB separation in combination with MALDI-TOF MS.

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				G. M. Fiedler, A. B. Leichtle, J. Kase, S. Baumann, U. Ceglarek, K. Felix, T. Conrad, H. Witzigmann, A. Weimann, C. Schutte, et al. Serum Peptidome Profiling Revealed Platelet Factor 4 as a Potential Discriminating Peptide Associated with Pancreatic Cancer Clin. Cancer Res., June 1, 2009; 15(11): 3812 - 3819. [Abstract] [Full Text] [PDF]

				S. Decramer, A. G. de Peredo, B. Breuil, H. Mischak, B. Monsarrat, J.-L. Bascands, and J. P. Schanstra Urine in Clinical Proteomics Mol. Cell. Proteomics, October 1, 2008; 7(10): 1850 - 1862. [Abstract] [Full Text] [PDF]