Home LiteratureArticle Details

PMID: 22706673 Published · epublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

A protease for 'middle-down' proteomics.

Nature methods ·Vol. 9 ·No. 8 ·2012-06-17 ·Pages 822-4

Wu C, Tran JC, Zamdborg L, Durbin KR, Li M, Ahlf DR, Early BP, Thomas PM, Sweedler JV, Kelleher NL

Abstract

We developed a method for restricted enzymatic proteolysis using the outer membrane protease T (OmpT) to produce large peptides (>6.3 kDa on average) for mass spectrometry-based proteomics. Using this approach to analyze prefractionated high-mass HeLa proteins, we identified 3,697 unique peptides from 1,038 proteins. We demonstrated the ability of large OmpT peptides to differentiate closely related protein isoforms and to enable the detection of many post-translational modifications.

MeSH Terms

HeLa Cells Humans Mass Spectrometry Peptides/analysis,chemistry,metabolism Protein Isoforms/chemistry,metabolism Protein Processing, Post-Translational Proteolysis Proteomics/methods Serine Endopeptidases/chemistry,metabolism

Chemicals

Peptides Protein Isoforms Serine Endopeptidases omptin outer membrane protease

Authors & Affiliations

10 authors, click to expand affiliations / ORCID

Wu Cong

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Tran John C

Zamdborg Leonid

Durbin Kenneth R

Li Mingxi

Ahlf Dorothy R

Early Bryan P

Thomas Paul M

Sweedler Jonathan V

Kelleher Neil L

References (24)

24 references, click to expand

Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.
Nat Methods. 2007 Mar;4(3):207-14 PMID: 17327847
Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.
Nature. 2008 Oct 30;455(7217):1251-4 PMID: 18820680
A novel activity of OmpT. Proteolysis under extreme denaturing conditions.
J Biol Chem. 1995 Jun 2;270(22):12990-4 PMID: 7768890
Toward efficient analysis of >70 kDa proteins with 100% sequence coverage.
Proteomics. 2001 Aug;1(8):927-33 PMID: 11683509
Mapping intact protein isoforms in discovery mode using top-down proteomics.
Nature. 2011 Oct 30;480(7376):254-8 PMID: 22037311
Informatics and multiplexing of intact protein identification in bacteria and the archaea.
Nat Biotechnol. 2001 Oct;19(10):952-7 PMID: 11581661
Interpretation of shotgun proteomic data: the protein inference problem.
Mol Cell Proteomics. 2005 Oct;4(10):1419-40 PMID: 16009968
Substrate specificity of the Escherichia coli outer membrane protease OmpT.
J Bacteriol. 2004 Sep;186(17):5919-25 PMID: 15317797
In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT.
Eur J Biochem. 2000 Feb;267(3):885-93 PMID: 10651827
A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.
Anal Biochem. 1984 Apr;138(1):141-3 PMID: 6731838
Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidase.
Nat Methods. 2008 May;5(5):405-7 PMID: 18425140
High-throughput middle-down analysis using an orbitrap.
J Proteome Res. 2010 Aug 6;9(8):3886-90 PMID: 20557100
Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site.
EMBO J. 2001 Sep 17;20(18):5033-9 PMID: 11566868
A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.
Mol Cell Proteomics. 2009 Dec;8(12):2759-69 PMID: 19828875
In vitro folding of Escherichia coli outer-membrane phospholipase A.
Eur J Biochem. 1995 Aug 15;232(1):214-9 PMID: 7556153
Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation.
Anal Chem. 2008 Mar 1;80(5):1568-73 PMID: 18229945
Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries.
Biochemistry. 2001 Feb 13;40(6):1694-701 PMID: 11327829
A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome.
J Am Soc Mass Spectrom. 2009 Dec;20(12):2183-91 PMID: 19747844
Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR).
J Proteome Res. 2005 Jul-Aug;4(4):1155-70 PMID: 16083266
Statistical significance for genomewide studies.
Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9440-5 PMID: 12883005
Chemistry. Mass spectrometry: bottom-up or top-down?
Science. 2006 Oct 6;314(5796):65-6 PMID: 17023639
Multiplexed size separation of intact proteins in solution phase for mass spectrometry.
Anal Chem. 2009 Aug 1;81(15):6201-9 PMID: 19572727
Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT.
Eur J Biochem. 2002 Mar;269(6):1746-52 PMID: 11895445
Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions.
Biosci Biotechnol Biochem. 2002 Jan;66(1):127-34 PMID: 11866094

Article Info

Journal

Nature methods

Abbr.

Nat Methods

ISSN

1548-7105

DOI

10.1038/nmeth.2074

Published

2012-06-17

Epub

2012-00-17

Pages

822-4

Language

English

Region

United States

NLM ID

101215604

PMCID

PMC3430368

Subset

Grants

NIDA NIH HHS · F30 DA026672 · United States

NIDA NIH HHS · P30 DA018310 · United States

NIGMS NIH HHS · R01 GM067193 · United States

External Links

PubMed source DOI: 10.1038/nmeth.2074 PMC Full Text View journal details

Analysis Services

A protease for 'middle-down' proteomics.

Contact

WeChat Official Account