Dataset Information

Leveraging immonium ions for identifying and targeting acyl-lysine modifications in proteomic datasets

ABSTRACT: Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify these modifications more confidently can help assess the scope of these modifications in large proteomic datasets. Herein we analyze the utility of acyl-lysine immonium ions for identifying the modifications in proteomic datasets. We demonstrate that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion strongly. Implementing an analysis that accounted for features within each MS2 spectra, this method allows peptides with short chain acyl-lysine modifications to be clearly identified in complex lysates. Immonium ions can also be used to validate novel acyl-modifications; in this study we report the first examples of 3-hydroxylpimelyl-lysine modification and validate them using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

TISSUE(S): Hela Cell

SUBMITTER: Janine Fu

LAB HEAD: Joseph A. Loo

PROVIDER: PXD018758 | Pride | 2020-09-09

REPOSITORIES: Pride

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Publications

Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets.

Muroski John M JM Fu Janine Y JY Nguyen Hong Hanh HH Ogorzalek Loo Rachel R RR Loo Joseph A JA

Proteomics 20200925 3-4

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared t ...[more]

PMID: 32896103

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Project description:Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using Acyl-ATP (AcATP) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Protein extracts were labelled with NHS-LC-Biotin, purified over neutravidin and eluted with formic acid. Eluted proteins were separated by 1-D SDS Gel and digested with Trypsin. Peptide mixtures were analysed by LC-MS/MS on an LTQ Velos. Spectra were searched using Mascot 2.3 using a fixed modification of cysteine (57.02 Da for carbamidomethyl), and variable modifications of methionine (15.99 Da for oxidation) and lysine (339.16 and 355.16 Da for BHAc and OBHAc labeling, respectively). A mass tolerance was set to 0.3 Da for the precursor ions and 0.4 Da for fragment ions. Up to two mis-cleavages for trypsin were permitted since the labeling would prevent cleavage at the labeled lysine. The MS2 spectra were searched against the TAIR10_pep_2012 (version November 2012) containing 35386 proteins of Arabidopsis thaliana, supplemented with a small database with 1095 artefact proteins and a reversed decoy database of the same proteins (totals 72962 entries). An oxidized version of the modifier was observed. Alternatively Arabidopsis leaf proteome was labeled with desthiobiotin-acyl ATP and digested with trypsin. Labeled peptides were purified on streptavidin matrix and sequenced by LC-MS/MS on an LTQ linear ion trap. Here the MS2 spectra were searched using SEQUEST against the TAIR9_pep_20090619 database (version June 2009) containing 32,769 proteins of Arabidopsis thaliana. MS2 searches included fixed iodoacetamide modification of cysteine (57 Da for alkylation), and variable modifications of methionine (16 Da for oxidation) and lysine (196 Da for DBAcATP labeling). Up to three mis-cleavages with trypsin were allowed and non-tryptic or half-tryptic peptides were excluded. A mass tolerance was set to 3 Da for the precursor ions and 1 Da for fragment ions.

Dataset Information

Leveraging immonium ions for identifying and targeting acyl-lysine modifications in proteomic datasets

Publications

Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets.

Similar Datasets

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