Abstract
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.
Highlights
The effectiveness of urine proteomics has been recognized year by year because urine reflects various biological phenomenon in our body [1]
To improve the efficiency of trypsin digestion, we focused on the dissolution of extracted urine proteins
We investigated the recovery rate and condition of precipitated urine protein to evaluate the ability of the four different kinds of precipitation method by following a study design described in Section 2.2 (Figure 1A)
Summary
The effectiveness of urine proteomics has been recognized year by year because urine reflects various biological phenomenon in our body [1]. Urine examination has some benefits to detect physiological changes and diseases in an individual. Blood examination shows internal environment clearly; it has a problem that the sampling itself is invasive and is difficult to repeat several times during the short term [2]. Many studies conducted examinations utilizing urine samples, and those results provided valuable information in various medical and health care fields [3]. Urine proteomics is actively performed to figure out the internal environment change in the human body. An investigation of biomarkers for various diseases is intensively performed using urine proteomics approach [4]. Throughout some studies, several biomarkers have already been discovered for the detection and prediction of diseases [5,6]
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