Proteomics

Dataset Information

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Comprehensive identification of crosslinked peptides from methyltransferase Hmt1 and its substrate Npl3: use of multiple crosslinkers, mass spectrometry approaches and software platforms


ABSTRACT: Chemical-Crosslinking Mass Spectrometry (XLMS) using MS-cleavable crosslinkers is fast becoming an established technique in the study of protein-protein interactions for both small and large scale samples. With the increased uptake of XLMS as a technique, different combinations of crosslinker types, fragmentation strategies and analysis programs are being applied to a diverse array of biological samples. This study is focused on understanding how the three variables of an XLMS experiment – crosslinker type, fragmentation and program – could generate differences in identifications, leading to increased biological coverage of a sample. Here we probe for the first time, the known enzyme:substrate interaction between yeast arginine methyltransferase Hmt1p and its substrate, heterologous nucleolar protein Npl3p. We use this known interaction as a platform to compare two analysis programs, MeroX and XlinkX2.0, using two crosslinker types, DSSO and DSBU, and two mass-spectrometry fragmentation strategies, CID/ETD and SteppedHCD. Through these we also compare different algorithm strategies, Precursor and Reporter-Ion, as well as assess the impact of restricting data searches to lysine only crosslinks versus the inclusion of serine, threonine and tyrosine as reactive residues. From this analysis we show direct evidence of Hmt1p in contact with its known methylation sites on Npl3p, the intrinsically disordered “SRGG” region. We also show through our multi-crosslinker, multi-fragmentation and multi-software approach that two approaches leads to greater understanding and depth of a crosslinked sample, and this is due to some combinations of experimental variables creating greater coverage of crosslinks than others.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Daniela-Lee Smith  

LAB HEAD: Marc R Wilkins

PROVIDER: PXD008348 | Pride | 2018-07-20

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
BuUrBU_R1_LPMI_CIDETD.csv Csv
BuUrBu_R1_Enumeration_CIDETD.csv Csv
BuUrBu_R1_Enumeration_SteppedHCD.csv Csv
BuUrBu_R1_LPMI_SteppedHCD.csv Csv
BuUrBu_R2_Enumeration_CIDETD.csv Csv
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Publications

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

Smith Daniela-Lee DL   Götze Michael M   Bartolec Tara K TK   Hart-Smith Gene G   Wilkins Marc R MR  

Analytical chemistry 20180725 15


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 interdependen  ...[more]

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