Biomedical Engineering Reference
In-Depth Information
Chapter 14
Protocol for Quantitative Proteomics of Cellular Membranes
and Membrane Rafts
Andrew J. Thompson and Ritchie Williamson
Abstract
Proteomic analysis of membrane and membrane raft proteins is complicated by their inherent insolubility,
which exacerbates difficulties with in-solution digestion of the proteins prior to ESI-LC-MS/MS. In-
gel digestion yields more comprehensive proteomic and protein coverage of membrane/membrane raft
samples, for example by LC-MS/MS of protein samples resolved by 1D SDS-polyacrylamide gel elec-
trophoresis. Although this type of analysis can be performed quantitatively by labelling at the protein
level, for instance by SILAC, the separation of proteins on a resolving gel complicates the application
of other quantitative methods that employ post-digestion labelling techniques. This chapter describes an
alternative protocol to prepare membrane or membrane raft protein samples to be isolated, but not sep-
arated, as unresolved bands in a gel. Focusing as a single band enables the confident excision of different
samples in their entirety, to be digested, labelled, and fractionated for quantitative mass spectrometric
analysis.
Key words:
Quantitative proteomics, membrane raft, Isobaric tagging, in-gel digestion, strong
cation exchange chromatography, LC-MS/MS.
1.
Introduction
Isobaric tagging for quantitative mass spectrometry has several
advantages over other quantitative MS-based methods including
multiplexed analysis of four to eight samples in a single experi-
ment and generation of quantitative information simultaneously
with peptide sequencing. Typical workflows involve differential
tagging of samples after in-solution digestion. However, this can
be problematic for membrane proteomics due to the insoluble
nature of the proteins, and the analysis of membrane rafts, which
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