Proteomics pp 83-98 | Cite as

High pH Reversed-Phase Micro-Columns for Simple, Sensitive, and Efficient Fractionation of Proteome and (TMT labeled) Phosphoproteome Digests

  • Benjamin Ruprecht
  • Jana Zecha
  • Daniel P. Zolg
  • Bernhard KusterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)


Despite recent advances in mass spectrometric sequencing speed and improved sensitivity, the in-depth analysis of proteomes still widely relies on off-line peptide separation and fractionation to deal with the enormous molecular complexity of shotgun digested proteomes. While a multitude of methods has been established for off-line peptide separation using HPLC columns, their use can be limited particularly when sample quantities are scarce. In this protocol, we describe an approach which combines high pH reversed-phase peptide separation into few fractions in StageTip micro-columns. This miniaturized sample preparation method enhances peptide recovery and hence improves sensitivity. This is particularly useful when working with limited sample amounts obtained from e.g., phosphopeptide enrichments or tissue biopsies. Essentially the same approach can also be applied for multiplexed analysis using tandem mass tags (TMT) and can be parallelized in order to deliver the required throughput. Here, we provide a step-by-step protocol for TMT6plex labeling of peptides, the construction of StageTips, sample fractionation and pooling schemes adjusted to different types of analytes, mass spectrometric sample measurement, and downstream data processing using MaxQuant. To illustrate the expected results using this protocol, we provide results from an unlabeled and a TMT6plex labeled phosphopeptide sample leading to the identification of >17,000 phosphopeptides in 8 h (Q Exactive HF) and >23,000 TMT6plex labeled phosphopeptides (Q Exactive Plus) in 12 h of measurement time. Importantly, this protocol is equally applicable to the fractionation of full proteome digests.

Key words

Fractionation Proteomics Sample preparation Mass spectrometry Phosphorylation Isotope labeling 





Acquisition gain control


Formic acid


Higher energy collision induced dissociation


High-performance liquid chromatography


Hydrophilic strong anion exchange


Immobilized metal ion affinity chromatography


Injection time


Mass spectrometer


Tandem mass spectrometry


Ammonium formate




Strong anion exchange


Strong cation exchange


Stop and go extraction tip


Triethylammonium bicarbonate


Trifluoroacetic acid


Titanium dioxide


Tandem mass tag


Parts per million


Peptide spectrum match


Phosphotyrosine, -serine, -threonine


Zwitterionic hydrophilic interaction liquid chromatography


  1. 1.
    Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509:582–587CrossRefPubMedGoogle Scholar
  2. 2.
    Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3:1630–1638CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gauci S, Helbig AO, Slijper M et al (2009) Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem 81:4493–4501CrossRefPubMedGoogle Scholar
  4. 4.
    McNulty DE, Annan RS (2008) Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics 7:971–980CrossRefPubMedGoogle Scholar
  5. 5.
    Ruprecht B, Koch H, Medard G et al (2015) Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns. Mol Cell Proteomics 14:205–215CrossRefPubMedGoogle Scholar
  6. 6.
    Batth TS, Francavilla C, Olsen JV (2014) Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J Proteome Res 13:6176–6186CrossRefPubMedGoogle Scholar
  7. 7.
    Kettenbach AN, Gerber SA (2011) Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal Chem 83:7635–7644CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ishihama Y, Rappsilber J, Mann M (2006) Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J Proteome Res 5:988–994CrossRefPubMedGoogle Scholar
  9. 9.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906CrossRefPubMedGoogle Scholar
  10. 10.
    Lawrence RT, Perez EM, Hernández D et al (2015) The proteomic landscape of triple-negative breast cancer. Cell Rep 11:630–644CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kitata RB, Dimayacyac-Esleta BRT, Choong W-K et al (2015) Mining missing membrane proteins by high-pH reverse-phase StageTip fractionation and multiple reaction monitoring mass spectrometry. J Proteome Res 14:3658–3669CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wiśniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res 8:5674–5678CrossRefPubMedGoogle Scholar
  13. 13.
    Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904CrossRefPubMedGoogle Scholar
  14. 14.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372CrossRefPubMedGoogle Scholar
  15. 15.
    Cox J, Neuhauser N, Michalski A et al (2011) Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res 10:1794–1805CrossRefPubMedGoogle Scholar
  16. 16.
    Olsen JV, Blagoev B, Gnad F et al (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648CrossRefPubMedGoogle Scholar
  17. 17.
    Winter D, Seidler J, Ziv Y et al (2009) Citrate boosts the performance of phosphopeptide analysis by UPLC-ESI-MS/MS. J Proteome Res 8:418–424CrossRefPubMedGoogle Scholar
  18. 18.
    Ow SY, Salim M, Noirel J et al (2009) iTRAQ underestimation in simple and complex mixtures: “the good, the bad and the ugly”. J Proteome Res 8:5347–5355CrossRefPubMedGoogle Scholar
  19. 19.
    Werner T, Sweetman G, Savitski MF et al (2014) Ion coalescence of neutron encoded TMT 10-plex reporter ions. Anal Chem 86:3594–3601CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Benjamin Ruprecht
    • 1
    • 2
  • Jana Zecha
    • 1
    • 3
    • 4
  • Daniel P. Zolg
    • 1
  • Bernhard Kuster
    • 1
    • 2
    • 3
    • 4
    • 5
    Email author
  1. 1.Technische Universität MünchenFreisingGermany
  2. 2.Center for Integrated Protein Science Munich (CIPSM)FreisingGermany
  3. 3.German Cancer Consortium (DKTK)HeidelbergGermany
  4. 4.German Cancer Research Center (DKFZ)HeidelbergGermany
  5. 5.Bavarian Bimolecular Mass Spectrometry CenterTechnische Universität MünchenFreisingGermany

Personalised recommendations