Lc ms ms orbitrap

Proteins were isolated and analyzed by slight modification of a previously described procedure (Brzychczy-Włoch et al., 2013c (link)). Briefly, bacteria were cultured on brain heart infusion broth (Biocorp) at 37°C for 24 h. After centrifugation, they were suspended directly in the buffer for electrophoresis according to Heilmann et al. (1996) (link). This made it possible to isolate moonlighting proteins and preserve their native structure. Protein concentration was analyzed using the Lowry’s method (Lowry et al., 1951 (link)). The immunoreactive proteins were identified by immunoblotting within a complex protein mixture that has been fractionated using discontinuous gradient polyacrylamide gel (5, 7.5, 10, and 12.5%). Individual reactive spots were cut out from gels and submitted to tryptic digestion, then analyzed by mass spectrometry (spectrometer LC-MS/MS Orbitrap, Thermo). Proteins were identified by Mascot¹ (Matrix Science, London, United Kingdom) and statistical methods.

After Western blot analysis, the immunoreactive proteins were separated and purified by preparative electrophoresis in denaturing condition (SDS-PAGE) using Prep-Cell apparatus (Model 491 Bio-Rad). Bands of interest were cut out and digested by trypsin (Roche) to obtain a mixture of peptides and analyzed by liquid chromatography (LC), where the mass fragments were measured using mass spectrometer LC-MS/MS Orbitrap (Thermo). Peptides were first trapped and desalted on the enrichment column (Zorbax 300SB-C18, 0.3 × 5 mm, Agilent) for 5 min (solvent: 2.5 % acetonitrile/0.5 % formic acid), then separated on a Zorbax 300SB-C18, 75 μm × 150 mm column (Agilent), using a linear gradient from 10 to 32 % B (solvent A: 5 % acetonitrile in water, solvent B: acetonitrile, both with 0.1 % formic acid). Ions of interest were data-dependently subjected to MS/MS, according to the expected charge state distribution of peptide ions. Proteins were identified by comparative analysis of peptides masses (NCBInr, UniProt, Bethesda, USA), using MS/MS ion search of the Mascot search engine (Matrix Science, London, UK, http://www.matrixscience.com/) and statistical analysis. Only peptide matches with a score of 1000 or above were accepted. Immunoreactive properties of separated proteins were re-tested using immunoblotting.

Protein identification was performed as previously described by us (Górska et al., 2016 (link)). Briefly, the immunoreactive protein were separated and purified using Prep-Cell apparatus (Model 491 Bio-Rad). Individual spots were cut out from gels and submitted to tryptic digestion, and analyzed by mass spectrometry (spectrometer LC-MS/MS Orbitrap, Thermo). Mascot (Matrix Science, London, UK, http://www.matrixscience.com) and statistical analysis were used to identify proteins from peptide mass fingerprints. All searches were performed against the database for Bifidobacterium species. Immunoreactivity of separated proteins was proved using immunoblotting.