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Challenges in plasma membrane phosphoproteomics - PubMed

Review

Challenges in plasma membrane phosphoproteomics

Benjamin C Orsburn et al. Expert Rev Proteomics. 2011 Aug.

Abstract

The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.

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Conflict of interest statement

Financial & competing interests disclosure The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1
Figure 1. Steps and possible approaches detailed in this review for a membrane phosphoproteomics experiment

CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CID: Collision-induced dissociation; CNBr: Cyanogen bromide; ECD:Electron capture dissociation; ETD: Electron transfer dissociation; FACE: Filter-based affinity capturing and elution; FASP: Filter-aided sample preparation; HCD: Higher-energy collisional dissociation; HILIC: Hydrophilic interaction chromatography; IEF: Isoelectric focusing; IMAC: Immobilized metal affinity chromatography; mAb: Monoclonal antibody; MS: Mass spectrometry; NHS: N-hydroxysuccinimide; NL: Neutral loss; SAX: Strong anion chromatography; SCX: Strong cation chromatography; SDS: Sodium dodecyl sulfate; TiO2: Titanium dioxide.

Figure 2
Figure 2. Overlapping fragmentation information obtained by using the different fragmentation methods available on our Orbitrap XL plus electron transfer dissociation to fragment a synthetic phosphopeptide (Waters, MA, USA), with a m+H+ of 1368.6776

The phosphorylated threonine residue is represented by ‘t’. (A) Collision-induced dissociation of the intact phosphopeptide. (B) ‘Neutral loss’ MS3 performed on the doubly charged fragment at 635.83, representing the loss of phosphoric acid from the threonine residue, represented by ‘T’. (C & D) Higher-energy collisional dissociation and electron transfer dissociation fragmentation of the intact phosphopeptide, respectively.

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