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

Abstract

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 enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for 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, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

“…A major limitation of this analysis strategy however, is that proteolytic digestion increases the complexity of the component mixture by up to several orders of magnitude, thereby placing significant demands on the performance of the analytical methodologies employed for peptide separation and analysis. Furthermore, although affinity purification [7] and off-and on-line multidimensional chromatographic [8] approaches have been developed for simplification of these complex peptide mixtures prior to MS, and dynamic exclusion methods [9,10] have been developed to allow greater numbers of distinct peptide ions to be selected for MS/MS analysis, information regarding the identity of proteins represented as low abundance peptides is commonly not obtained, thereby limiting the dynamic range [11].…”

Differential polypeptide display: the search for the elusive target

“…Wang and Dass [73] developed a method for the analysis of bioactive peptides in bovine adrenal medulla, utilizing a combination of fast-HPLC and ESI-MS, also using non-porous silica-based C18 columns. Several peptides, including the opioid peptides methionine-enkephalin (Met-Enk) and leucine-enkephalin (Leu-Enk), were identified with MS-MS. An elegant approach to identify urinary polypeptides was published by Spahr et al [74]. The authors used HPLC coupled to MS-MS to analyze tryptic digests of pooled human urinary proteins and identified more than 100 polypeptides.…”

Advances in analytical mass spectrometry to improve screening for inherited metabolic diseases

“…to assess molecular fingerprints of CSF proteins as new diagnostic links to acute and chronic neurometabolic CNS disease [55]. High resolution urinary proteome analysis was recently shown being capable to identify 124 gene products out of 1,450 peptide MS/MS spectra in less than the time required for classical 2-dimensional gel electrophoresis [61]. Such new techniques have great potential to supplement existing urinary screening for metabolic disorders by providing diagnostic protein and peptide information.…”