Dataset Information

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

ABSTRACT: Identification and characterization of Tryp-N, A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli

SUBMITTER: Jonathan Ipsaro

LAB HEAD: Darryl J. Pappin

PROVIDER: PXD017048 | Pride | 2020-04-22

REPOSITORIES: Pride

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Publications

Tryp-N: A Thermostable Protease for the Production of N-terminal Argininyl and Lysinyl Peptides.

Wilson John P JP Ipsaro Jonathan J JJ Del Giudice Samantha N SN Turna Nikita Saha NS Gauss Carla M CM Dusenbury Katharine H KH Marquart Krisann K Rivera Keith D KD Pappin Darryl J DJ

Journal of proteome research 20200320 4

Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic ...[more]

PMID: 32141294

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Project description:The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.

			Action	DRS
	Ad.dat	Other
	Ad.raw	Raw
	As.dat	Other
	As.raw	Raw
	Ct.dat	Other

Dataset Information

Tryp-N: A thermostable protease for the production of N-terminal argininyl and lysinyl peptides

Dataset's files

Publications

Tryp-N: A Thermostable Protease for the Production of N-terminal Argininyl and Lysinyl Peptides.

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