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.
Project description:We have explored the use of electrostatic repulsion hydrophilic interaction chromatography (ERLIC) as an alternative to the gold-standard in shotgun proteomics: reversed-phase (RP) LC for online ESI-MS/MS. Conditions for sample solubilization and initial gradient conditions were optimized to strike a balance between peptide solubility and maximum peptide retention when using mobile phase with high organic solvent concentration. Online ERLIC-MS demonstrated a 57% increase in total peptide identifications compared to RP-MS. We examined the mechanism of this improved performance and found that it stems from ERLIC's propensity to retain longer peptides, which can be identified with greater confidence. Online nanoscale ERLIC-MS provides a powerful new tool for enhancing MS-based shotgun proteomic in a broad range of applications.
Project description:The ribosomal L3 protein was identified as a novel target in linezolid (LZD)-resistant Mycobacterium tuberculosis strains. Next-generation sequencing confirmed rplC T460C as the sole mutation in an LZD-resistant M. tuberculosis H37Rv strain selected in vitro. Sequencing analysis revealed the rplC T460C mutation in eight further LZD-resistant isolates (three in vitro-selected mutants and five patient isolates, including isolates from three different patients that developed LZD resistance during treatment) but in none of the susceptible control strains (n = 84).
Project description:One of the HIV-1 vaccine design efforts has focused on developing a recombinant HIV-1 trimeric envelope glycoprotein (Env) as an immunogen to induce broadly neutralizing antibodies. A native-like immunogen, the BG505.DS.SOSIP.664 gp140 (Env) construct has been well-characterized as a vaccine candidate. This vaccine candidate comprises of three identical gp120 and truncated gp41 subunits that form into a trimer of heterodimers. During production, recombinant Env is expressed as a gp140 precursor polypeptide in which a furin cleavable site is engineered to generate a heterodimer of gp120 and gp41 subunits. Each heterodimer is connected by an intermolecular disulfide bond, and three heterodimers form into a trimer. Furin cleavage is an important factor to mimic native-like HIV-1 Env conformations and is needed to help induce an immune response. Therefore, it is critical to monitor cleavage for ensuring functionality of the Env vaccine product. In this paper, a new RPLC-UV method coupled with reduction was developed to routinely determine the percentage of uncleaved gp140 relative to the cleaved gp120 and gp41 subunits. Baseline separation was achieved among the gp120, gp41 and uncleaved gp140 peaks, thus enabling relative quantification of uncleaved gp140. Overall, this RPLC-UV approach has been successfully applied to support Env vaccine candidate developments.