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The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

Biofluid Biomarker Center, Niigata University, Niigata 9502181, Japan
Division of Clinical Nephrology and Rheumatology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata 9518510, Japan
Department of Pediatrics, National Hospital Organization Niigata Hospital, Kashiwazaki 9458585, Japan
Life Science Research Laboratory, Tosoh Corporation, Kanagawa 252-1123, Japan
Department of Clinical laboratory, Shinrakuen Hospital, Niigata 9502087, Japan
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Methods Protoc. 2019, 2(2), 46;
Received: 17 April 2019 / Revised: 22 May 2019 / Accepted: 4 June 2019 / Published: 7 June 2019
The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery. View Full-Text
Keywords: Urine proteomics; Urine protein precipitation; Urine biomarker Urine proteomics; Urine protein precipitation; Urine biomarker
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Saito, S.; Hirao, Y.; Quadery, A.F.; Xu, B.; Elguoshy, A.; Fujinaka, H.; Koma, S.; Yamamoto, K.; Yamamoto, T. The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics. Methods Protoc. 2019, 2, 46.

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