磷酸蛋白质组学
化学
蛋白质组学
样品制备
色谱法
定量蛋白质组学
生物化学
蛋白质磷酸化
蛋白激酶A
酶
基因
作者
Fujia Wang,Tim S. Veth,Marije E. Kuipers,Maarten Altelaar,Kelly E. Stecker
标识
DOI:10.1021/acs.analchem.3c00324
摘要
A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in large-scale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.
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