Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

Hart‐Smith, Gene; Low, Jason K. K.; Erce, Melissa A.; Wilkins, Marc R.

doi:10.1007/s13361-012-0417-8

Cited by 35 publications

(46 citation statements)

References 52 publications

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“…LTQ Orbitrap Pro ETD analyses were performed as above, with the following exception: precursor ions were fragmented via ETD rather than CID, using parameters described previously (32).…”

Section: Methodsmentioning

confidence: 99%

“…Where relevant, spectra were also inspected for the presence of fragment ions associated with neutral losses diagnostic for arginine methylation (32,34).…”

Section: Methodsmentioning

confidence: 99%

“…1B, methylpeptides associated with previously reported S. cerevisiae methylation sites, specifically methylation sites annotated in Uniprot or additional literature sources (10,14,16,17,30,32,(41)(42)(43), were identified in unfiltered sequence database search outputs and tracked.…”

Section: Fig 1 Workflows Employed In the Investigation Of Methyl-psmentioning

confidence: 99%

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Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Hart‐Smith

Yagoub

Tay

et al. 2016

Molecular & Cellular Proteomics

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All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13 CD 3 -methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methylPSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-␤-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl- Post-translational methylation is a widespread protein modification, which predominantly occurs on lysine and arginine residues (1). Protein-lysine methyltransferases catalyze the methylation of lysine residues; these enzymes facilitate the incorporation of methyl groups into the N atoms of lysine residues to produce either mono-, di-, or tri-methyllysine (MML, 1 DML, and TML, respectively). Protein-arginine methyltransferases catalyze the methylation of arginine residues; these enzymes primarily act upon N G atoms to produce mono, asymmetric di-, or symmetric di-methylarginine, although the enzyme-mediated modification of N ␦ atoms to produce ␦-MMA has also been reported in Saccharomyces cerevisiae (2).Traditionally, lysine and arginine methylation have been closely associated with histone proteins, and their crucial roles in modifying chromatin structure have been extensively studied (3). In recent years, however, a growing number of large scale methylation site discovery experiments have indiFrom the ‡New South

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“…LTQ Orbitrap Pro ETD analyses were performed as above, with the following exception: precursor ions were fragmented via ETD rather than CID, using parameters described previously (32).…”

Section: Methodsmentioning

confidence: 99%

“…Where relevant, spectra were also inspected for the presence of fragment ions associated with neutral losses diagnostic for arginine methylation (32,34).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Hart‐Smith

Yagoub

Tay

et al. 2016

Molecular & Cellular Proteomics

Self Cite

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“…Verification of Methylation-The methylation states of the proteins were verified via Western blotting (14), and methylated arginine residues were confirmed using electron-transfer dissociation (ETD) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (20). Where appropriate, proteins were purified via his-tag purification before ETD LC-MS/MS.…”

Section: Construction Of C2h Vectors Carrying Interacting Proteins Andmentioning

confidence: 99%

“…MS and ETD MS/MS were performed using an LTQ Orbitrap Velos (Thermo Electron, Bremen, Germany) hybrid linear ion trap and Orbitrap mass spectrometer using previously described experimental conditions (14,20). The precursor ions were measured in the Oribitrap and the ETD fragments were measured in the linear trap.…”

Section: Construction Of C2h Vectors Carrying Interacting Proteins Andmentioning

confidence: 99%

Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

Erce

Abeygunawardena

Low

et al. 2013

Molecular & Cellular Proteomics

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Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated. Molecular & Cellular

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The activity of a yeast Family 16 methyltransferase, Efm2, is affected by a conserved tryptophan and its N‐terminal region

et al. 2016

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The Family 16 methyltransferases are a group of eukaryotic nonhistone protein methyltransferases. Sixteen of these have recently been described in yeast and human, but little is known about their sequence and structural features. Here we investigate one of these methyltransferases, Saccharomyces cerevisiae elongation factor methyltransferase 2 (Efm2), by site‐directed mutagenesis and truncation. We show that an active site‐associated tryptophan, invariant in Family 16 methyltransferases and at position 222 in Efm2, is important for methyltransferase activity. A second highly conserved tryptophan, at position 318 in Efm2, is likely involved in S‐adenosyl methionine binding but is of lesser consequence for catalysis. By truncation analysis, we show that the N‐terminal 50–200 amino acids of Efm2 are critical for its methyltransferase activity. As N‐terminal regions are variable among Family 16 methyltransferases, this suggests a possible role in determining substrate specificity. This is consistent with recently solved structures that show the core of Family 16 methyltransferases to be near‐identical but the N termini to be structurally quite different. Finally, we show that Efm2 can exist as an oligomer but that its N terminus is not necessary for oligomerisation to occur.

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Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

Cited by 35 publications

References 52 publications

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates

Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

The activity of a yeast Family 16 methyltransferase, Efm2, is affected by a conserved tryptophan and its N‐terminal region

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