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Methods for the Isolation of Phosphoproteins and Phosphopeptides for Mass Spectrometry Analysis: Toward Increased Functional Phosphoproteomics

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Sample Preparation in Biological Mass Spectrometry

Abstract

Reversible phosphorylation is arguably the best understood, if not the most important mechanism a cell employs to dynamically regulate protein (and thereby cellular) activity. In recent years the field of proteomics has acquired an impressive tool kit with which to study protein phosphorylation on a more global scale. These tools build upon classical techniques and incorporate novel methodologies, all propelled by rapid advances in mass spectrometers that function with increased speed, sensitivity and resolution. The depth and breadth of phosphoproteomic analyses are becoming increasingly comprehensive from both cumulative and single-study perspectives. Phosphoproteomics is now poised to become increasingly functional as experimental design incorporates the amassed wealth of current biological understanding with novel technologies and quantitative mass spectrometry. In this chapter, three important phosphoproteomic sample preparation methodologies are described: classical phospho-dependent affinity chromatography, tandem strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC), and immunoaffinity chromatography. Each method is discussed with an eye toward designing experiments that maximize phospho-dependent functional understanding.

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Abbreviations

AQUA:

Absolute Quantification

ApoER2:

ApoE Receptor 2

ATP:

Adenosine Triphosphate

ATM:

Ataxia-Telangiectasia Mutated

ATR:

Ataxia-Telangiectasia Mutated and Rad3-Related

BRCT:

Breast Cancer 1 C-terminal

CST:

Cell Signaling Technology

Crk:

CT10-Regulator of Kinase

CrkL:

CT10-Regulator of Kinase-Like

Dab1:

Disabled-1

FHA:

Forkhead-Associated

GST:

Glutathione-S Transferase

IMAC:

Immobilized Metal Affinity Chromatography

LC:

Liquid Chromatography

MH2:

Mother Against Decapentaplegic Homology 2

MS:

Mass Spectrometry

PhPO4 :

Phenyl Phosphate

PI 3-K:

Phosphoinositide 3-Kinase

PLC:

Phospholipase C

PTB:

Phosphotyrosine-Binding

pY-Dab1:

Phosphotyrosyl-Disabled-1

PTM:

Posttranslational Modification

RTK:

Receptor Tyrosine Kinase

RSK:

90 Kilodalton Ribosomal S6 Kinase

Sf9:

Spodoptera frugiperda-9

SFK:

Src Family Tyrosine Kinase

SDS-PAGE:

Sodium Dodecyl Dulfate Polyacrylamide Gel Electrophoresis

SH2:

Src Homology 2

SCX:

Strong Cation Exchange Chromatography

TCA:

Trichloroacetic Acid

VLDLR:

Very Low Density Lipoprotein Receptor

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Acknowledgements

We thank Lionel Arnaud who was a critical collaborator in the Cooper lab during the development and execution of the methodology to identify pY-Dab1-interacting proteins. We thank Jon Cooper (Fred Hutchinson Cancer Research Center) for support and discussions as well as additional members from his laboratory, particularly Abir Mukherjee, Tara Herrick and Priscilla Kronstad-O’Brien. We thank Steve Gygi (Harvard Medical School) for support and discussions, and members of his laboratory for the same, in particular, Scott Gerber, Wilhelm Haas and Sean Beausoleil. Judit Villén and Xue Li from the Gygi lab played an important role in the initial characterization and development of the SCX-IMAC methods presented here. We thank Steve Elledge (Harvard Medical School) and two members from his laboratory, Shuhei Matsuoka and Chunshui Zhou. Shuhei performed the anti-Akt phospho-substrate peptide IP from tryptic peptides and Chunshui gave helpful insights during the development of the SCX-IMAC methods and provided the tryptic yeast peptides. We thank John Blenis (Harvard Medical School) and two members of his lab, Philippe Roux and Jessie Hanrahan who assisted in the preparation of the cells for the Anti-Akt phoshpho-substrate peptide IP from chymotryptic peptides. We thank John Rush (Cell Signaling Technology) for supplying the AQUA reference peptides. The authors were supported primarily by the following grants during the work presented here and during the writing of this manuscript: NIH 5T32CA09657; NIH HG00041; NSF IOS 1021795; Vermont Genetics Network and NIH/NCRR P20RR16462.

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Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA

    Tapasree Goswami

  2. Department of Biology and Vermont Genetics Network Proteomics Facility, University of Vermont, Burlington, VT, 05405, USA

    Bryan A. Ballif

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  1. , Department of Genetics and Complex Disea, Harvard School of Public Health, Harvard, Huntington Ave., SPH-1 665, Boston, 02115, Massachusetts, USA

    Alexander R. Ivanov

  2. , Research and Development, Pressure BioSciences, Inc., Norfolk Ave. 14, South Easton, 02375, Massachusetts, USA

    Alexander V. Lazarev

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Goswami, T., Ballif, B.A. (2011). Methods for the Isolation of Phosphoproteins and Phosphopeptides for Mass Spectrometry Analysis: Toward Increased Functional Phosphoproteomics. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_29

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