Characterization of the Interaction between Arginine Methyltransferase Hmt1 and Its Substrate Npl3: Use of Multiple Cross-Linkers, Mass Spectrometric Approaches, and Software Platforms

• Published in: Analytical chemistry (Washington) 2018-08, Vol.90 (15), p.9101-9108
• Main Authors: Smith, Daniela-Lee, Götze, Michael, Bartolec, Tara K, Hart-Smith, Gene, Wilkins, Marc R
• Format: Article
• Language: English
• Subjects: Chemistry; Crosslinking; DNA methylation; Electron transfer reactions; Energy of dissociation; Eukaryotes; Mass spectrometry; Organic chemistry; Peptides; Proteins; Saccharomyces cerevisiae; Spectroscopy; Substrates; Yeast
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.8b01525

This study investigated the enzyme–substrate interaction between Saccharomyces cerevisiae arginine methyltransferase Hmt1p and nucleolar protein Npl3p, using chemical cross linking/mass spectrometry (XL/MS). We show that XL/MS can capture transient interprotein interactions that occur during the process of methylation, involving a disordered region in Npl3p with tandem SRGG repeats, and we confirm that Hmt1p and Npl3p exist as homomultimers. Additionally, the study investigated the interdependencies between variables of an XL/MS experiment that lead to the identification of identical or different cross-linked peptides. We report that there are substantial benefits, in terms of biologically relevant cross-links identified, that result from the use of two mass-spectrometry-cleavable cross-linkers [disuccinimido sulfoxide (DSSO) and disuccinimido dibutyric urea (DSBU)], two fragmentation approaches [collision-induced dissociation and electron-transfer dissociation (CID+ETD)] and stepped high-energy collision dissociation (HCD)], and two programs (MeroX and XlinkX). We also show that there are specific combinations of XL/MS methods that are more successful than others for the two proteins investigated here; these are explored in detail in the text. Data are available via ProteomeXchange with identifier PXD008348.