Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides
ABSTRACT CID has become a routine method for fragmentation of peptides in shotgun proteomics, whereas electron transfer dissociation (ETD) has been described as a preferred method for peptides carrying labile PTMs. Though both of these fragmentation techniques have their obvious advantages, they also have their own drawbacks. By combining data from CID and ETD fragmentation, some of these disadvantages can potentially be overcome because of the complementarity of fragment ions produced. To evaluate alternating CID and ETD fragmentation, we analyzed a complex mixture of phosphopeptides on an LTQ-Orbitrap mass spectrometer. When the CID and ETD-derived spectra were searched separately, we observed 2504, 491, 2584, and 3249 phosphopeptide-spectrum matches from CID alone, ETD alone, decision tree-based CID/ETD, and alternating CID and ETD, respectively. Combining CID and ETD spectra prior to database searching should, intuitively, be superior to either method alone. However, when spectra from the alternating CID and ETD method were merged prior to database searching, we observed a reduction in the number of phosphopeptide-spectrum matches. The poorer identification rates observed after merging CID and ETD spectra are a reflection of a lack of optimized search algorithms for carrying out such searches and perhaps inherent weaknesses of this approach. Thus, although alternating CID and ETD experiments for phosphopeptide identification are desirable for increasing the confidence of identifications, merging spectra prior to database search has to be carefully evaluated further in the context of the various algorithms before adopting it as a routine strategy.
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ABSTRACT: Analysis of the phosphoproteome by mass spectrometry has become a key technology for the characterisation of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable sidechain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or 'localise' the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.Proteomics 03/2015; 15(5-6). DOI:10.1002/pmic.201400372 · 3.97 Impact Factor
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ABSTRACT: Multi-enzyme digestion for protein sequencing and characterization by CID/ETD.•Simultaneous use of trypsin/chymotrypsin for the maximization of sequence.•Identification of PTMs, sequence variants and species-specific residues.•Increase of accuracy in sequence assignments by orthogonal fragmentation techniques.Analytica Chimica Acta 11/2014; DOI:10.1016/j.aca.2014.10.053 · 4.52 Impact Factor
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ABSTRACT: O-GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago and its significance has been implicated in several disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically challenging to characterize comprehensively and quantitatively because of its low abundance, low stoichiometry and extremely labile nature under conventional collision-induced dissociation tandem MS conditions. Herein, we review the recent advances addressing these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, including specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification and quantitative analysis of O-GlcNAcylation.Bioanalysis 10/2014; 6(19):2571-80. DOI:10.4155/bio.14.239 · 3.03 Impact Factor