Peptide Identification by Tandem Mass Spectrometry with Alternate Fragmentation Modes

Guthals, Adrian; Bandeira, Nuno

doi:10.1074/mcp.r112.018556

Cited by 67 publications

(59 citation statements)

References 92 publications

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“…MS instruments may implement collision-induced diffusion (CID), or may have electron capture/transfer dissociation or high energy collision dissociation for the fragmentation of peptides for MS/MS. These alternative fragmentation modes can result in significant improvement in the detection of PTM (Guthals & Bandeira, 2012). Enrichment of peptides containing certain PTM can also be applied before data acquisition.…”

Section: Highly Sensitive Methods For Lc-tandem-msmentioning

confidence: 99%

The Role of Casein Kinase I in the Drosophila Circadian Clock

Price

Fan

Keightley

et al. 2015

Circadian Rhythms and Biological Clocks, Part A

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Section: Highly Sensitive Methods For Lc-tandem-msmentioning

confidence: 99%

The Role of Casein Kinase I in the Drosophila Circadian Clock

Price

Fan

Keightley

et al. 2015

Circadian Rhythms and Biological Clocks, Part A

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“…The fraction of peptide backbone breaks, defined by the presence of a b or y fragment ion corresponding to a break in the peptide backbone, was calculated according to Ref. 33.…”

Section: Nano-lc Electrospray Ionization Ms/ms Of Hprp-fractionatedmentioning

confidence: 99%

Expanding Proteome Coverage with Orthogonal-specificity α-Lytic Proteases

Meyer

Kim

Maltby

et al. 2014

Molecular & Cellular Proteomics

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Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin's may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type ␣-lytic protease (WaLP) and an active site mutant of WaLP, M190A ␣-lytic protease (MaLP). We assess several relevant factors, including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. When data from separate digestions with trypsin, LysC, WaLP, and MaLP were combined, proteome coverage was increased by 101% relative to that achieved with trypsin digestion alone. To demonstrate how the gained sequence coverage can yield additional posttranslational modification information, we show the identification of a number of novel phosphorylation sites in the Schizosaccharomyces pombe proteome and include an illustrative example from the protein MPD2 wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined. Molecular & Cellular Proteomics 13: 10.1074/ mcp.M113.034710, 823-835, 2014.The most powerful technique for system-scale protein measurement, or proteomics, is mass-spectrometry-based proteomics (1). Although great progress has enabled the quantification of nearly all proteins expressed in yeast (2, 3), sequence coverage is often dismal, with some proteins being identified by a single peptide sequence. Complete amino acid coverage is valuable for comprehensive profiling of post-translational modifications (e.g. phosphorylation) and for quantification of splice variants. Low observed proteome coverage can be caused by several factors, including the wide dynamic range of protein concentrations in biological samples, splice variants, and unanticipated or unconsidered post-translational modifications (PTMs).

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“…While the generating function described here supports only unmodified peptides, it can be extended to analyze modified peptides by considering modified amino acid mass edges [as shown before (Kim et al, 2010)]. Further improvements are foreseeable with additional support for high-resolution MS/MS peak masses and incorporation of alternative fragmentation modes (e.g., HCD, ETD) to improve of the quality of PRM spectra, especially if from highly charged precursors (Guthals and Bandeira, 2012). Given that MS-GFDB supports multiple fragmentation modes and that we utilize PepNovo + to transform MS/MS spectra to PRM spectra, it is possible for this approach to support any fragmentation mode since PepNovo + can be trained to process new types of spectra (Guthals et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

The Generating Function Approach for Peptide Identification in Spectral Networks

Guthals

Boucher

Bandeira

2014

Lecture Notes in Computer Science

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Tandem mass (MS/MS) spectrometry has become the method of choice for protein identification and has launched a quest for the identification of every translated protein and peptide. However, computational developments have lagged behind the pace of modern data acquisition protocols and have become a major bottleneck in proteomics analysis of complex samples. As it stands today, attempts to identify MS/MS spectra against large databases (e.g., the human microbiome or 6-frame translation of the human genome) face a search space that is 10-100 times larger than the human proteome, where it becomes increasingly challenging to separate between true and false peptide matches. As a result, the sensitivity of current state-of-the-art database search methods drops by nearly 38% to such low identification rates that almost 90% of all MS/MS spectra are left as unidentified. We address this problem by extending the generating function approach to rigorously compute the joint spectral probability of multiple spectra being matched to peptides with overlapping sequences, thus enabling the confident assignment of higher significance to overlapping peptide-spectrum matches (PSMs). We find that these joint spectral probabilities can be several orders of magnitude more significant than individual PSMs, even in the ideal case when perfect separation between signal and noise peaks could be achieved per individual MS/MS spectrum. After benchmarking this approach on a typical lysate MS/MS dataset, we show that the proposed intersecting spectral probabilities for spectra from overlapping peptides improve peptide identification by 30-62%.

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Peptide Identification by Tandem Mass Spectrometry with Alternate Fragmentation Modes

Cited by 67 publications

References 92 publications

The Role of Casein Kinase I in the Drosophila Circadian Clock

The Role of Casein Kinase I in the Drosophila Circadian Clock

Expanding Proteome Coverage with Orthogonal-specificity α-Lytic Proteases

The Generating Function Approach for Peptide Identification in Spectral Networks

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