The Enzyme Effect: Broadening the Horizon of MS Optimization to Nontryptic Digestion in Proteomics

IF 3.1 2区 化学 Q2 BIOCHEMICAL RESEARCH METHODS
Kinga Nagy, Péter Sándor, Károly Vékey, László Drahos and Ágnes Révész*, 
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引用次数: 0

Abstract

In recent years, alternative enzymes with varied specificities have gained importance in MS-based bottom-up proteomics, offering orthogonal information about biological samples and advantages in certain applications. However, most mass spectrometric workflows are optimized for tryptic digests. This raises the questions of whether enzyme specificity impacts mass spectrometry and if current methods for nontryptic digests are suboptimal. The success of peptide and protein identifications relies on the information content of MS/MS spectra, influenced by collision energy in collision-induced dissociation. We investigated this by conducting LC-MS/MS measurements with different enzymes, including trypsin, Arg-C, Glu-C, Asp-N, and chymotrypsin, at varying collision energies. We analyzed peptide scores for thousands of peptides and determined optimal collision energy (CE) values. Our results showed a linear m/z dependence for all enzymes, with Glu-C, Asp-N, and chymotrypsin requiring significantly lower energies than trypsin and Arg-C. We proposed a tailored CE selection method for these alternative enzymes, applying ca. 20% lower energy compared to tryptic peptides. This would result in a 10–15 eV decrease on a Bruker QTof instrument and a 5–6 NCE% (normalized collision energy) difference on an Orbitrap. The optimized method improved bottom-up proteomics performance by 8–32%, as measured by peptide identification and sequence coverage. The different trends in fragmentation behavior were linked to the effects of C-terminal basic amino acids for Arg-C and trypsin, stabilizing y fragment ions. This optimized method boosts the performance and provides insight into the impact of enzyme specificity. Data sets are available in the MassIVE repository (MSV000095066).

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来源期刊
CiteScore
5.50
自引率
9.40%
发文量
257
审稿时长
1 months
期刊介绍: The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role. Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives
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