Discovering the N-Terminal Methylome by Repurposing of Proteomic Datasets

Abstract

Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-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, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-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 first position. We tested three potentially methylated proteins and confirmed the α-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 that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.

The protein methylation network in yeast: A landmark in completeness for a eukaryotic post-translational modification

“…Two ribosomal proteins which contain this motif, Rps25 and Rpl12, are known to be methylated by Ntm1 ( 33 ). Two further proteins, Rpt1 and Hsp31, have been shown to be methylated at their N-termini which contain this motif ( 34 , 35 ) and are therefore likely Ntm1 substrates. Beyond this, there are a further six proteins whose N-termini match the recognition motif of Ntm1 and who colocalize with Ntm1 in the cytoplasm—Afg2, Art10, Lsg1, New1, Ola1, and Tma46; these may be additional Ntm1 substrates.…”

Structure, Activity and Function of the Dual Protein Lysine and Protein N-Terminal Methyltransferase METTL13

“…The Nt of proteins is most frequently acetylated [24]; only in rare occasions subject to methylation [25]. Notably, the major Nt acetyltransferase A complex (NatA) is reported to acetylate substrates with a small amino acid such as Gly, Ala or Ser in the second position, and after excision of the initiator Met residue [26].…”

Multicomponent Oxidative Nitrile Thiazolidination Reaction for Selective Modification of N-terminal Dimethylation Posttranslational Modification

“…Although α-N-terminal dimethylation (Nme 2 ) posttranslational modification (PTM) has been discovered four decades ago, − it recently gained significant attention due to its emerging role in regulating various biological processes, including DNA repair, epigenetics, translation fidelity, mitosis, genome stability, and its implications in numerous human disorders such as cancer, inflammation, neurodegenerative, and cardiovascular disorders. − Moreover, previous studies with eukaryotes showed that the N-terminus methylation takes place at the conserved canonical sequence A/S-PK, but recent studies with prokaryotes and humans showed several non-canonical sequences that are methylated at the N-terminus, thus supporting that Nme 2 is a widespread PTM. − In contrast to dimethyl lysine (Kme 2 ) PTM, Nme 2 is largely underexplored due to the lack of affinity reagents and antibodies for its identification. The current method to identify Nme 2 PTM involves the use of mass spectrometry (MS) but there are several challenges to its accurate identification by MS; , (i) low natural abundance of Nme 2 PTM in complex mixtures; (ii) lack of affinity agents to selectively enrich Nme 2 PTMs; (iii) change in mass by two methyl groups (28 Da) on the N-terminus is identical to the N-formylation and Kme 2 PTMs (28 Da) and identical to the mass difference between Ala Vs Val (28 Da), leading to the false identification. So far, only ∼300 N-methylation sites (including mono-, di-, and tri- N -methyl states) have been discovered despite evidence of their vast existence in varying species. − To completely understand the role of Nme 2 PTM, its global identification is required which is possible by a pan-selective chemical method for labeling Nme 2 sites in a proteome.…”

“…12−14 In contrast to dimethyl lysine (Kme 2 ) PTM, Nme 2 is largely underexplored due to the lack of affinity reagents and antibodies for its identification. The current method to identify Nme 2 PTM involves the use of mass spectrometry (MS) but there are several challenges to its accurate identification by MS; 15,16 (i) low natural abundance of Nme 2 PTM in complex mixtures; (ii) lack of affinity agents to selectively enrich Nme 2 PTMs; (iii) change in mass by two methyl groups (28 Da) on the N-terminus is identical to the N-formylation and Kme 2 PTMs (28 Da) and identical to the mass difference between Ala Vs Val (28 Da), leading to the false identification. So far, only ∼300 N-methylation sites (including mono-, di-, and tri-N-methyl states) have been discovered despite evidence of their vast existence in varying species.…”

“…(b) Selective enrichment of Nme 2 proteins from the cell lysate followed by proteomic analysis on enriched and digested proteins or on bead digestion of proteins. The proteomic analysis of enriched and digested proteins showed capturing of 1394 Nme 2 proteins as compared to the negative control(15). Top 5 most abundant Nme 2modified proteins obtained after enrichment were listed.…”