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

Abstract

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 (Mr, 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.