Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I. Profiling an unfractionated tryptic digest

The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS /MS). The entire urinary protein mixture was denatured, reduced and enzymat ically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-o f-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture wa s analyzed four separate times, with the mass spectrometer selecting ions f or fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unat tended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and t ime for these translations or expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a s ingle two-dimensional gel.