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Proteomics pp 47–60Cite as

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography

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

Abstract

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

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Abbreviations

ACN:

Acetonitrile

AGC:

Acquisition gain control

CAA:

Chloroacetamide

DTT:

Dithiothreitol

FA:

Formic acid

FCS:

Fetal calf serum

HCD:

Higher energy collision induced dissociation

HCl:

Hydrochloride

HPLC:

High-performance liquid chromatography

I.D.:

Inner diameter

IMAC:

Immobilized metal ion affinity chromatography

MeOH:

Methanol

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

PBS:

Phosphate buffered saline

Ppm:

parts per million

PSM:

Peptide spectrum match

pY/pS/Pt:

Phosphotyrosine, -serine, -threonine

TFA:

Trifluoro acetic acid

TiO2 :

Titanium dioxide

Tris:

Tris(hydroxymethyl)aminomethane

v/v:

Volume/volume

w/w:

Weight/weight

ZrO2 :

Zirconium dioxide

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Author information

Authors and Affiliations

  1. Chair of Proteomics and Bioanalytics, Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany

    Benjamin Ruprecht, Heiner Koch, Martin Frejno, Bernhard Kuster & Simone Lemeer

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

    Benjamin Ruprecht, Bernhard Kuster & Simone Lemeer

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

    Heiner Koch & Bernhard Kuster

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

    Heiner Koch & Bernhard Kuster

  5. Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic

    Petra Domasinska

  6. Faculty of Chemical Technology, Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic

    Petra Domasinska

  7. Department of Oncology, University of Oxford, Oxford, UK

    Martin Frejno

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

    Bernhard Kuster

  9. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Simone Lemeer

Authors
  1. Benjamin Ruprecht
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  2. Heiner Koch
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  3. Petra Domasinska
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  4. Martin Frejno
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  5. Bernhard Kuster
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  6. Simone Lemeer
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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., Koch, H., Domasinska, P., Frejno, M., Kuster, B., Lemeer, S. (2017). Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography. 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_5

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  • DOI: https://doi.org/10.1007/978-1-4939-6747-6_5

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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

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