Many trials for biomarker discovery have been going on by mass spectrometry (MS) in plasma, tissue, urine or others. Now a day, technologies of MS are still developing to provide new machines and softwares. In fact, a number of proteins and peptides identified with high confidence by MS have increased a lot during these several decades.
The urine remains as one of the preferable biological samples for clinical examinations for diagnosis or monitoring of diseases, due to its non-invasive nature of collection. Some proteins and peptides, which are smaller than albumin, may be secreted in the urine from plasma through the glomerulus in the kidney. To find biomarkers for systemic organs in the urine, we aimed to develop a workflow to identify the urinary small proteins and native peptides, which had been processed in the body and thought to be novel biomarkers for many diseases. However, there are several difficulties for this such as less amounts of the small proteins and native peptides, no established protocol for collection and purification of these proteins and peptide, no qualitative and quantitative methods by MS.
In this study, we focused on mass spectrometry of the urinary small proteins and native peptides and established new protocols to propose a workflow for biomarker discovery by peptidomics. By our workflow more than 10,000 distinct peptides have been identified. These peptides were grouped as 500 peptide clusters; C-peptide cluster includes its intact form and also peptides forms truncated at the N- or C-terminal.
In conclusion, identification of the small proteins and peptides is still challenging due to no standard protocols for sample preparation, MS analysis methods. We proposed a new workflow for MS-based identification of native peptides in the urine to promote biomarker discovery in the near future.