PXD000078

PXD000078 is an original dataset announced via ProteomeXchange.

Title	'Life and death' of extracytoplasmic proteins
Description	Gram-positive bacteria are known to export many proteins to the cell wall and growth medium and, accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed at determining the fate of native exported proteins in the model organism Bacillus subtilis. Specifically, we employed a GeLC-MS approach to distinguish the roles of the membrane-associated quality control proteases HtrA and HtrB from those of eight other proteases that are present in the cell wall and/or growth medium of B. subtilis. Notably, HtrA and HtrB were previously shown to counteract potentially detrimental 'protein export stresses' upon overproduction of membrane- or secreted proteins. Our results show that many secreted proteins, lipoproteins and membrane proteins of B. subtilis are potential substrates of extracytoplasmic proteases. Moreover, potentially important roles of HtrA and HtrB in the folding of native secreted proteins into a protease-resistant conformation, the liberation of lipoproteins from the membrane-cell wall interface, and the degradation of membrane proteins are uncovered. Altogether, our observations show that HtrA and HtrB are crucial for maintaining the integrity of the B. subtilis cell even under non-stress conditions. Bionformatics pipeline and data processing: MS and MS/MS data were acquired with the LTQ-Orbitrap mass spectrometer. After a survey scan in the Orbitrap (r= 30,000), MS/MS data were recorded for the five most intensive precursor ions in the linear ion trap. Singly charged ions were not taken into account for MS/MS analysis. The lock mass option was enabled throughout the analysis. The mass spectrometric data was then subjected to database searching via Sorcerer-Sequest. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version v.27, rev. 11). Sequest was set up to search a B. subtilis _target-decoy protein sequence database that included the complete proteome set of _B. subtilis _extracted from UniprotKB release 12.7, and a set of common laboratory contaminants compiled with Bioworks Browser (Thermo Fisher Scientific), assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a search tolerance of 10 ppm for the overview scans. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version Scaffold_3_00_04, Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they exceeded Xcorr values of 2.2, 3.5, 3.75 for doubly, triply and quadruply charged ions. A protein was regarded as significantly identified when at least two peptides per protein were identified in at least three of four biological replicates. These criteria resulted in no false positive identifications.
HostingRepository	PRIDE
AnnounceDate	2015-04-21
AnnouncementXML	Submission_2015-04-21_07:34:07.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Andreas Otto
SpeciesList	scientific name: Bacillus subtilis subsp. subtilis str. 168; NCBI TaxID: 224308;
ModificationList	monohydroxylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2012-11-28 07:09:08	ID requested
1	2014-10-15 07:27:24	announced
⏵ 2	2015-04-21 07:34:09	announced	Updated project metadata.

Krishnappa L, Dreisbach A, Otto A, Goosens VJ, Cranenburgh RM, Harwood CR, Becher D, van Dijl JM, Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis. J Proteome Res, 12(9):4101-10(2013) [pubmed]

curator keyword: Biological

submitter keyword: Bacillus subtilis, extracytoplasmic proteases, protein secretion, lipoprotein, membrane protein

Andreas Otto
contact affiliation	Institute for Microbiology
contact email	andreas.otto@uni-greifswald.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2014/10/PXD000078

PRIDE project URI

• PRIDE
  1. PXD000078
     1. Label: PRIDE project
     2. Name: 'Life and death' of extracytoplasmic proteins