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bioRxiv · 10.1101/2021.04.14.439552

Discovering the N-terminal Methylome by Repurposing of Proteomic Datasets

Chen, P.·Sobreira, T. J. P.·Hall, M. C.·Hazbun, T.
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Abstract

Protein -N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free -amino group at protein N-termini. To systematically explore the extent of -N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the -N-terminal methylome. This repurposing approach found evidence of -N-methylation of established and novel protein substrates with canonical N-terminal motifs of established -N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, -N-methylation of non-canonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of -N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation, and methylation, on amino acids at the 1st position. We tested three potentially methylated proteins and confirmed the -N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating proteomic searching for -N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global -N-terminal methylome investigations. The raw MS data that supports the findings of this study were deposited with PRIDE identifier: PXD022833. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/439552v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@124e5b9org.highwire.dtl.DTLVardef@1660eb7org.highwire.dtl.DTLVardef@15027aaorg.highwire.dtl.DTLVardef@15c0da6_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract (For TOC only). C_FIG

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Chen, P., Sobreira, T. J. P., Hall, M. C., Hazbun, T.. 2021-04-15. Discovering the N-terminal Methylome by Repurposing of Proteomic Datasets. https://doi.org/10.1101/2021.04.14.439552

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