Dataset Information

Triflic acid-treatment enables LC-MS/MS analysis of insoluble bacterial biomass

ABSTRACT: Established bacterial proteome sample preparations, including sonication and bead beating, leave insoluble carbohydrate-rich cell envelope pellets with an abundance of vital proteins often overlooked or missed in LC-MS/MS analyses. Triflic acid selectively removes glycans and we demonstrate that in comparison to sonication alone, incubation of whole bacterial cells as well as post-sonication insoluble pellets yields membrane and cell envelope-associated proteins for LC-MS/MS detection. We provide a detailed side-by-side comparison of triflic acid and sonication preparations for Gram- (Pseudomonas aeruginosa), Gram+ (Bacillus subtilis), and a complex bacterial human distal gut microbiome sample. Further, human Jurkat cells that lack a peptidoglycan and are readily solubilized by established methods, reveal only subtle differences in measurable proteins by LC-MS/MS between sonication and triflic acid preparations. Critically, we show that our new triflic acid-based proteome preparation method is broadly applicable and greatly improves our ability to detect and quantitate bacterial cell envelope proteins.

INSTRUMENT(S): LTQ, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Pseudomonas Aeruginosa Pao1 Bacillus Subtilis

SUBMITTER: Ana Wang

LAB HEAD: Dennis Wolan

PROVIDER: PXD009004 | Pride | 2019-01-29

REPOSITORIES: Pride

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Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass.

Wang Ana Y AY Thuy-Boun Peter S PS Stupp Gregory S GS Su Andrew I AI Wolan Dennis W DW

Journal of proteome research 20180808 9

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble postlysis fractions differ significantly. Additionally, in the case of Gram ...[more]

PMID: 30019906

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