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

@article{citeulike:1414978, 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 (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.}, address = {Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany.}, author = {Fiedler, G. M. and Baumann, S. and Leichtle, A. and Oltmann, A. and Kase, J. and Thiery, J. and Ceglarek, U. }, citeulike-article-id = {1414978}, doi = {10.1373/clinchem.2006.077834}, issn = {0009-9147}, journal = {Clin Chem}, keywords = {proteomics, urine}, month = {March}, number = {3}, pages = {421--428}, posted-at = {2007-06-27 07:37:29}, priority = {2}, title = {Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.}, url = {http://dx.doi.org/10.1373/clinchem.2006.077834}, volume = {53}, year = {2007} }

TY - JOUR ID - citeulike:1414978 L3 - citeulike-article-id:1414978 TI - Standardized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. JF - Clin Chem VL - 53 IS - 3 SP - 421 EP - 428 AD - Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany. SN - 0009-9147 N2 - 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. KW - proteomics KW - urine AU - Fiedler, GM AU - Baumann, S AU - Leichtle, A AU - Oltmann, A AU - Kase, J AU - Thiery, J AU - Ceglarek, U PY - 2007/03// UR - http://dx.doi.org/10.1373/clinchem.2006.077834 ER -