Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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 microg. 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.