Characterization of the interaction between arginine methyltransferase Hmt1 and its substrate Npl3: use of multiple cross-linkers, mass spectrometric approaches, and software platforms

Daniela Lee Smith, Michael Götze, Tara K. Bartolec, Gene Hart-Smith, Marc R. Wilkins*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)9101-9108
Number of pages8
JournalAnalytical Chemistry
Volume90
Issue number15
DOIs
Publication statusPublished - 7 Aug 2018
Externally publishedYes

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