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

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Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1550))

Abstract

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.

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Abbreviations

ACN:

Acetonitrile

AGC:

Acquisition gain control

CAA:

Chloroacetamide

DTT:

Dithiothreitol

FA:

Formic acid

FDR:

False discovery rate

HCl:

Hydrochloric acid

HPLC:

High-performance liquid chromatography

hSAX:

Hydrophilic strong anion exchange

IMAC:

Immobilized metal ion affinity chromatography

IT:

Injection time

MeOH:

Methanol

MS:

Mass spectrometer

MS/MS:

Tandem mass spectrometry

PBS:

Phosphate buffered saline

PSM:

Peptide spectrum match

RP:

Reversed-phase

SAX:

Strong anion exchange

SCX:

Strong cation exchange StageTip stop and go extraction tip

TCEP:

Tris-(2-carboxyethyl)-phosphin

TFA:

Trifluoroacetic acid

Tris:

Tris(hydroxymethyl)aminomethane v/v volume/volume

w/w:

Weight/weight

ZIC-HILIC:

Zwitterionic hydrophilic interaction liquid chromatography

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Author information

Authors and Affiliations

  1. Chair of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany

    Benjamin Ruprecht, Dongxue Wang & Bernhard Kuster

  2. Center for Integrated Protein Science Munich (CIPSM), Freising, Germany

    Benjamin Ruprecht & Bernhard Kuster

  3. Department of Chemistry, Sapienza Università di Roma, Rome, Italy

    Riccardo Zenezini Chiozzi

  4. Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan

    Li-Hua Li

  5. OmicScouts GmbH, Freising, Germany

    Hannes Hahne

  6. German Cancer Consortium (DKTK), Heidelberg, Germany

    Bernhard Kuster

  7. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Bernhard Kuster

  8. Bavarian Biomolecular Mass Spectrometry Center, Technische Universität München, Freising, Germany

    Bernhard Kuster

Authors
  1. Benjamin Ruprecht
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  2. Dongxue Wang
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  3. Riccardo Zenezini Chiozzi
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  4. Li-Hua Li
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  5. Hannes Hahne
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  6. Bernhard Kuster
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Corresponding author

Correspondence to Bernhard Kuster .

Editor information

Editors and Affiliations

  1. Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Lucio Comai

  2. Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Jonathan E. Katz

  3. Stanford University, Palo Alto, California, USA

    Parag Mallick

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Ruprecht, B., Wang, D., Chiozzi, R.Z., Li, LH., Hahne, H., Kuster, B. (2017). Hydrophilic Strong Anion Exchange (hSAX) Chromatography Enables Deep Fractionation of Tissue Proteomes. In: Comai, L., Katz, J., Mallick, P. (eds) Proteomics. Methods in Molecular Biology, vol 1550. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6747-6_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6747-6_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6745-2

  • Online ISBN: 978-1-4939-6747-6

  • eBook Packages: Springer Protocols

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