Defeating Major Contaminants in Fe³⁺- Immobilized Metal Ion Affinity Chromatography (IMAC) Phosphopeptide Enrichment.

ABSTRACT: Here we demonstrate that biomolecular contaminants, such as nucleic acid molecules, can seriously interfere with immobilized metal ion affinity chromatography (IMAC)-based phosphopeptide enrichments. We address and largely solve this issue, developing a robust protocol implementing methanol/chloroform protein precipitation and enzymatic digestion using benzonase, which degrades all forms of DNA and RNA, before IMAC-column loading. This simple procedure resulted in a drastic increase of enrichment sensitivity, enabling the identification of around 17,000 unique phosphopeptides and 12,500 unambiguously localized phosphosites in human cell-lines from a single LC-MS/MS run, constituting a 50% increase when compared with the standard protocol. The improved protocol was also applied to bacterial

SUBMITTER: Potel CM 

PROVIDER: S-EPMC5930410 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature