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PMID: 22498708 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients.

Nature protocols ·Vol. 7 ·No. 5 ·2012-04-12 ·Pages 882-90

Köcher T, Pichler P, Swart R, Mechtler K

Abstract

The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLC-MS/MS measurement time.

MeSH Terms
Cell Extracts/chemistry Chromatography, Liquid/methods HeLa Cells Humans Proteins/chemistry Proteomics/methods Tandem Mass Spectrometry/methods
Chemicals
Cell Extracts Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Köcher Thomas
Research Institute of Molecular Pathology, Vienna, Austria. [email protected]
Pichler Peter
Swart Remco
Mechtler Karl
References (38)
38 references, click to expand
  1. 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
  2. The ABC's (and XYZ's) of peptide sequencing.
    Nat Rev Mol Cell Biol. 2004 Sep;5(9):699-711 PMID: 15340378
  3. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.
    Nature. 2008 Oct 30;455(7217):1251-4 PMID: 18820680
  4. Higher-energy C-trap dissociation for peptide modification analysis.
    Nat Methods. 2007 Sep;4(9):709-12 PMID: 17721543
  5. Target profiling of small molecules by chemical proteomics.
    Nat Chem Biol. 2009 Sep;5(9):616-24 PMID: 19690537
  6. More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS.
    J Proteome Res. 2011 Apr 1;10(4):1785-93 PMID: 21309581
  7. Principles of electrospray ionization.
    Mol Cell Proteomics. 2011 Jul;10(7):M111.009407 PMID: 21742801
  8. An improved method for tracking and reducing the void volume in nano HPLC-MS with micro trapping columns.
    Anal Bioanal Chem. 2003 Aug;376(7):946-51 PMID: 12856097
  9. Systems biology of innate immunity.
    Immunol Rev. 2009 Jan;227(1):264-82 PMID: 19120490
  10. High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics.
    Anal Chem. 2002 Aug 15;74(16):4235-49 PMID: 12199598
  11. Sample preparation and digestion for proteomic analyses using spin filters.
    Proteomics. 2005 May;5(7):1742-5 PMID: 15761957
  12. Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics.
    Anal Chem. 2005 May 15;77(10):3090-100 PMID: 15889897
  13. Trypsin cleaves exclusively C-terminal to arginine and lysine residues.
    Mol Cell Proteomics. 2004 Jun;3(6):608-14 PMID: 15034119
  14. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.
    Anal Chem. 2002 Oct 15;74(20):5383-92 PMID: 12403597
  15. Taming the isobaric tagging elephant in the room in quantitative proteomics.
    Nat Methods. 2011 Oct 28;8(11):911-3 PMID: 22036744
  16. Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.
    Electrophoresis. 2000 May;21(9):1635-50 PMID: 10870950
  17. A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed.
    Mol Cell Proteomics. 2009 Dec;8(12):2759-69 PMID: 19828875
  18. Multidimensional LC separations in shotgun proteomics.
    Anal Chem. 2008 Oct 1;80(19):7187-93 PMID: 18826178
  19. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.
    Nat Biotechnol. 2001 Mar;19(3):242-7 PMID: 11231557
  20. Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.
    Mol Cell Proteomics. 2011 Aug;10(8):M110.003699 PMID: 21586754
  21. Reporting protein identification data: the next generation of guidelines.
    Mol Cell Proteomics. 2006 May;5(5):787-8 PMID: 16670253
  22. Analysis and validation of proteomic data generated by tandem mass spectrometry.
    Nat Methods. 2007 Oct;4(10):787-97 PMID: 17901868
  23. Preventing carryover of peptides and proteins in nano LC-MS separations.
    Anal Chem. 2009 Jul 15;81(14):5955-60 PMID: 19537771
  24. Mass spectrometry-based functional proteomics: from molecular machines to protein networks.
    Nat Methods. 2007 Oct;4(10):807-15 PMID: 17901870
  25. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.
    Mol Cell Proteomics. 2005 Dec;4(12):2010-21 PMID: 16249172
  26. Functional organization of the yeast proteome by systematic analysis of protein complexes.
    Nature. 2002 Jan 10;415(6868):141-7 PMID: 11805826
  27. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
    Nat Protoc. 2007;2(8):1896-906 PMID: 17703201
  28. A guided tour of the Trans-Proteomic Pipeline.
    Proteomics. 2010 Mar;10(6):1150-9 PMID: 20101611
  29. Analytical properties of the nanoelectrospray ion source.
    Anal Chem. 1996 Jan 1;68(1):1-8 PMID: 8779426
  30. Quality control in LC-MS/MS.
    Proteomics. 2011 Mar;11(6):1026-30 PMID: 21360669
  31. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.
    Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9528-33 PMID: 15210983
  32. A statistical model for identifying proteins by tandem mass spectrometry.
    Anal Chem. 2003 Sep 1;75(17):4646-58 PMID: 14632076
  33. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.
    Nat Protoc. 2009;4(5):698-705 PMID: 19373234
  34. The impact of systems approaches on biological problems in drug discovery.
    Nat Biotechnol. 2004 Oct;22(10):1215-7 PMID: 15470453
  35. Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides.
    Anal Chem. 2011 Apr 1;83(7):2699-704 PMID: 21388192
  36. Applying mass spectrometry-based proteomics to genetics, genomics and network biology.
    Nat Rev Genet. 2009 Sep;10(9):617-27 PMID: 19687803
  37. Mass spectrometry for proteomics.
    Curr Opin Chem Biol. 2008 Oct;12(5):483-90 PMID: 18718552
  38. False discovery rates of protein identifications: a strike against the two-peptide rule.
    J Proteome Res. 2009 Sep;8(9):4173-81 PMID: 19627159
Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2012.036
Email
[email protected]
Published
2012-04-12
Epub
2012-00-12
Pages
882-90
Language
English
Region
England
NLM ID
101284307
Subset
IM
External Links
PubMed source DOI: 10.1038/nprot.2012.036 View journal details
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