Simultaneous Quantification of the Acetylome and Succinylome by One-Pot Affinity Enrichment

ABSTRACT: Protein post-translational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here we describe a streamlined, quantitative label-free proteomic workflow – “one-pot” PTM enrichment – which enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein. Coupled with a label-free, data-independent acquisition (DIA), we identified and quantified 2235 acetylated and 2173 succinylated peptides with the one-pot method and show that peak areas are highly correlated between the one-pot and traditional single-PTM enrichments. The ‘one-pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and we show that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Birgit Schilling  

PROVIDER: MSV000081906 | MassIVE | Tue Jan 09 10:30:00 GMT 2018

SECONDARY ACCESSION(S): PXD008640

REPOSITORIES: MassIVE