Project description:The studied compound SN12 significantly inhibited biofilm formation in Pseudomonas aeruginosa, but its regulatory mechanism is unknown. To determine the mechanism of action, TMT tagging was used to analyse the proteomics of P. aeruginosa PAO1. Three groups of blank controls treated with PBS were compared with three groups of experimental groups treated with 1 μM compound to assess its effect. Protein expression was identified and analysed using RPLC-MS technology subsequent to the extraction of the entire proteome.
Project description:Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom-up proteomics. It has approached 4000-8000 protein identifications (IDs) from a human cell line, mouse brains or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least five hundred micrograms of complex proteome digests were required for the LC-CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom-up proteomics of mass-limited samples. In this work, we coupled micro-scale reversed-phase LC (µRPLC) based peptide prefractionation to dynamic pH junction based CZE-MS/MS for deep bottom-up proteomics of the MCF7 breast cancer cell proteome starting with only 5-µg peptides. The dynamic pH junction based CZE enabled a 500-nL sample injection from as low as a 1.5-µL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of µRPLC prefractionation were investigated, C18 ZipTip and nanoflow RPLC. C18 ZipTip-CZE-MS/MS identified 4453 proteins from 5 µg of the MCF7 proteome digest and showed good qualitative and quantitative reproducibility. Nanoflow RPLC-CZE-MS/MS produced over 7500 protein IDs and nearly 60000 peptide IDs from the 5-µg MCF7 proteome digest. The nanoflow RPLC-CZE-MS/MS platform reduced the required amount of complex proteome digests for LC-CZE-MS/MS-based deep bottom-up proteomics by two orders of magnitude. Our work provides the proteomicscommunity with a powerful tool for deep and highly sensitive proteomics.
Project description:These five raw files represent five technical replicate RPLC data-dependent acquisition analyses of the peptides produced through trypsin digestion and DTT/iodoacetamide reduction/alkylation of an FFPE human colon tumor. The data, collected on a Q-Exactive, offer high resolution for both precursor and fragment ions. Lisa Zimmerman collected these data.