Assessment of ultrasonic-assisted enzymatic digestion of complex protein mixtures by high-resolution mass spectrometry

Abstract

Background & Objectives Large-scale proteome analysis by mass spectrometry is commonly preceded by enzymatic digestion of proteins. The conventional protocol for in-solution digest of complex protein mixtures includes trypsin and is performed at room temperature for at least 12h. To improve this time-consuming method we assessed the efficiency of focused ultrasound-assisted enzymatic digest. It has previously been shown that ultrasonication can dramatically reduce digestion time. However, it is not known how this affects protein identification and quantification when applied to complex protein mixtures such as whole cell lysates. Here, we explore in-depth the proteome of embryonic stem cells comparing trypsin-digestion methods with and without ultrasound support side-by-side. Methods In-solution protein digest Protein extracts were digested with sequencing-grade trypsin (Promega) for 1, 5, 10, 20, 30, 40min and 1h in the presence or absence of focused ultrasonication (Covaris). A conventional digestion overnight was included. Reactions were stopped by acidification and heat. Peptides were desalted by reverse phase C18 StageTips. Mass spectrometry Peptides were analyzed on a QExactive (Thermo Scientific) coupled to Easy nLC 1000 (Proxeon). Raw files were processed by MaxQuant v1.3.0.3 software. Peptide searches were performed by Andromeda search engine against the UniProt database. Results and Conclusions Focused ultrasonication clearly enhanced the enzymatic digestion and can be implemented in the daily workflow of mass spectrometry-based proteome analysis.