Dataset Information

A rapid methods development workflow for high-throughput quantitative proteomic applications

ABSTRACT: we developed a workflow using data acquired from discovery proteomic experiments, retention time prediction, and standard-flow chromatography to rapidly develop targeted proteomic assays

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J petzold

PROVIDER: PXD011212 | Pride | 2019-06-27

REPOSITORIES: Pride

ACCESS DATA

Publications

A rapid methods development workflow for high-throughput quantitative proteomic applications.

Chen Yan Y Vu Jonathan J Thompson Mitchell G MG Sharpless William A WA Chan Leanne Jade G LJG Gin Jennifer W JW Keasling Jay D JD Adams Paul D PD Petzold Christopher J CJ

PloS one 20190214 2

Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven significant progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Yet, these system-wide experiments are typically the starting point for hypothesis-driven, fo ...[more]

PMID: 30763335

Similar Datasets

Project description:MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a cysteine protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Dataset Information

A rapid methods development workflow for high-throughput quantitative proteomic applications

Publications

A rapid methods development workflow for high-throughput quantitative proteomic applications.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure