Proteomics pp 69-82 | Cite as

Hydrophilic Strong Anion Exchange (hSAX) Chromatography Enables Deep Fractionation of Tissue Proteomes

  • Benjamin Ruprecht
  • Dongxue Wang
  • Riccardo Zenezini Chiozzi
  • Li-Hua Li
  • Hannes Hahne
  • Bernhard KusterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)


The bottom-up proteomic analysis of cell line and tissue samples to a depth > 10,000 proteins still represents a considerable challenge because of the sheer number of peptides generated by proteolytic digestions and the high dynamic range of protein expression. As a result, comprehensive protein coverage requires multidimensional peptide separation. Recently, off-line hydrophilic strong cation exchange (hSAX) chromatography has proven its merits for high resolution separation of peptides due to its high degree of orthogonality to reversed-phase liquid chromatography. Here we describe the use of hSAX for the deep analysis of tissue proteomes. The protocol includes optimized sample preparation steps (lysis with the aid of mechanical disruption, one-step disulfide bridge reduction and alkylation), setup and operation of hSAX columns and gradients, desalting of hSAX fractions prior to LC-MS/MS analysis, and suggestions for the choice of data acquisition parameters and data analysis using MaxQuant. Application of the protocol to the fractionation of 300 μg human brain tissue digest led to the identification of more than 100,000 unique peptide sequences representing over 10,195 proteins and 9,500 genes in 3 days of measurement time on a Q Exactive Plus mass spectrometer.

Key words

Proteomics Deep fractionation Chromatography Strong anion exchange Tissue proteomics 





Acquisition gain control






Formic acid


False discovery rate


Hydrochloric acid


High-performance liquid chromatography


Hydrophilic strong anion exchange


Immobilized metal ion affinity chromatography


Injection time




Mass spectrometer


Tandem mass spectrometry


Phosphate buffered saline


Peptide spectrum match




Strong anion exchange


Strong cation exchange StageTip stop and go extraction tip




Trifluoroacetic acid


Tris(hydroxymethyl)aminomethane v/v volume/volume




Zwitterionic hydrophilic interaction liquid chromatography


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Benjamin Ruprecht
    • 1
    • 2
  • Dongxue Wang
    • 1
  • Riccardo Zenezini Chiozzi
    • 3
  • Li-Hua Li
    • 4
  • Hannes Hahne
    • 5
  • Bernhard Kuster
    • 1
    • 2
    • 6
    • 7
    • 8
    Email author
  1. 1.Chair of Proteomics and Bioanalytics, Technische Universität MünchenFreisingGermany
  2. 2.Center for Integrated Protein Science Munich (CIPSM)FreisingGermany
  3. 3.Department of ChemistrySapienza Università di RomaRomeItaly
  4. 4.Department of Pathology and Laboratory MedicineTaipei Veterans General HospitalTaipeiTaiwan
  5. 5.OmicScouts GmbHFreisingGermany
  6. 6.German Cancer Consortium (DKTK)HeidelbergGermany
  7. 7.German Cancer Research Center (DKFZ)HeidelbergGermany
  8. 8.Bavarian Biomolecular Mass Spectrometry CenterTechnische Universität MünchenFreisingGermany

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