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

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.

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

“…11 and Source Data File). Such quantities are easily obtained from biological sources and the number of identifications is comparable to figures reported in the recent nano-flow LC literature [37][38][39] . In contrast to nano-flow LC, the micro-flow LC system showed no obvious sign of overloading even when injecting as much as 20 µg protein digest of non-depleted plasma protein digest, ( Supplementary Fig.…”

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica—molecular background revealed by quantitative proteomics

“…The supernatant was withdrawn, and the obtained protein pellets were air-dried. The protein pellet was resuspended in sterile 8 M urea/50 mM Tris–HCl/50 mM EDTA buffer (pH 8.0), according to Saito et al ( 2019 ). The protein profile was examined for the protein integrity by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970 ).…”

miniTurbo‐based interactomics of two plasma membrane‐localized SNARE proteins in Marchantia polymorpha

“…Previously published studies using TurboID and miniTurbo identified the depletion of free biotin after labelling as a critical step for pulldown of biotinylated proteins using streptavidin beads (Mair et al ., 2019; Zhang et al ., 2019, 2021; Arora et al ., 2020). We therefore tested three different approaches, methanol : chloroform precipitation, PD‐10 gravity column desalting and Zeba spin column desalting, to remove excess free biotin from M. polymorpha cell extracts (Mair et al ., 2019; Saito et al ., 2019; Zhang et al ., 2019). Among these three methods, the methanol : chloroform precipitation approach has not been tested for free biotin depletion from plant materials.…”