single-pot solid-phase-enhanced sample preparation
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The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-μg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
The tegument, as the surface layer of adult male and female Schistosoma spp. represents the protective barrier of the worms to the hostile environment of the host bloodstream. Here we present the first comparative analysis of sex-specific tegument proteins of paired or virgin Schistosoma mansoni. We applied a new and highly sensitive workflow, allowing detection of even low abundance proteins. Therefore, a streptavidin-biotin affinity purification technique in combination with single pot solid-phase enhanced sample preparation was established for subsequent LC-MS/MS analysis. We were able to identify 1519 tegument proteins for male and female virgin and paired worms and categorized them by sex. Bioinformatic analysis revealed an involvement of female-specific tegument proteins in signaling pathways of cellular processes and antioxidant mechanisms. Male-specific proteins were found to be enriched in processes linked to phosphorylation and signal transduction. This suggests a task sharing between the sexes that might be necessary for survival in the host. Our datasets provide a basis for further studies to understand and ultimately decipher the strategies of the two worm sexes to evade the immune system.
- MeSH
- chromatografie kapalinová MeSH
- proteiny červů metabolismus MeSH
- proteom * metabolismus MeSH
- Schistosoma mansoni * metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
