Using in-solution digestion, peptide fractionation, and a Q exactive mass spectrometer to analyze the proteome of clathrin-coated vesicles

Georg H H Borner; Andrew B Fielding

doi:10.1101/pdb.prot084137

Using in-solution digestion, peptide fractionation, and a Q exactive mass spectrometer to analyze the proteome of clathrin-coated vesicles

Cold Spring Harb Protoc. 2014 Nov 3;2014(11):1192-5. doi: 10.1101/pdb.prot084137.

Authors

Georg H H Borner¹, Andrew B Fielding²

Affiliations

¹ Department of Proteomics and Signal Transduction, Max Plank Institute of Biochemistry, 82152 Martinsried, Germany;
² Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, United Kingdom.

PMID: 25368314
DOI: 10.1101/pdb.prot084137

Abstract

The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by in-solution proteolytic digest and subsequent peptide fractionation, followed by analysis in a Q Exactive mass spectrometer.

MeSH terms

Cell Fractionation / methods*
Clathrin-Coated Vesicles / chemistry*
HeLa Cells
Humans
Mass Spectrometry / methods*
Proteolysis
Proteome / analysis*

Substances

Proteome