Terminal proteomics: N‐ and C‐terminal analyses for high‐fidelity identification of proteins using MS

Nakazawa, Takashi; Yamaguchi, Minoru; Okamura, Taka‐aki; Ando, Eiji; Nishimura, Osamu; Tsunasawa, Susumu

doi:10.1002/pmic.200700084

Cited by 46 publications

(30 citation statements)

References 64 publications

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“…Perhaps the most challenging portions of the toxins to sequence are the termini, where fragmentation is frequently not observed between the terminal and penultimate residues. Here, it may prove necessary to apply additional derivatization strategies or C-terminal digestion strategies that enhance detection of the terminal residues (44,45). Another longstanding mass spectrometric sequencing problem is the ambiguity between the isobaric pair Leu and Ile.…”

Section: Discussionmentioning

confidence: 99%

Rapid sensitive analysis of cysteine rich peptide venom components

Ueberheide

Fenyö

Alewood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

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Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).conotoxins ͉ cysteine derivatization ͉ de novo sequencing ͉ electron transfer dissociation ͉ mass spectrometry V enomous animals such as spiders, snakes, scorpions, and certain sea snails produce a vast array of bioactive peptides, many of which are rich in disulfide bonds that stabilize their 3-dimensional structures. The enormous size of this natural combinatorial library can be appreciated by considering just the Ϸ500 known species of cone snail, which are estimated to produce Ͼ50,000 distinct peptide toxins (conotoxins) (1, 2); and this number is dwarfed by the toxins present in the Ϸ38,000 known species of spider (3). Interest in these peptide toxins derives in part from their potent and specific interactions with ion channels (3, 4) and in part from their structural integrity as disulfide-rich miniproteins (5, 6) Consequently they have become important tools for studying ion channels (7) and have considerable potential as pharmaceuticals (8, 9).One limit on the utilization of this rich resource of bioactive peptide toxins has been the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and in certain cases (e.g., cone snail venom) can contain extensive posttranslational modifications. The extraordinary complexity of crude venom samples (often containing Ͼ100 components) (4) and the lack of DNA databases for many of...

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Section: Discussionmentioning

confidence: 99%

Rapid sensitive analysis of cysteine rich peptide venom components

Ueberheide

Fenyö

Alewood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

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“…It is impossible to cover with this section all the possible problems deriving from MS/MS sequencing procedures, which are well described in many tutorials and reviews [72,73]. MS/MS is a very powerful method for sequencing peptides, but due to strong fragmentation dependence on peptide structure, de novo sequencing is often difficult.…”

Section: Ms/msmentioning

confidence: 99%

Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us?

Messana

Inzitari

Fanali

et al. 2008

J of Separation Science

131

140

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This review briefly depicts several salient points of the current status of knowledge on salivary peptidoma. It outlines the intrinsic difficulties in its characterization connected to different factors of variability, such as: i) the high genetic polymorphisms, complicated by individual insertions/deletions and alternative splicing; ii) complex post-translational maturations comprehending different proteolytic cleavages, glycosylation, phosphorylation and sulfation processes; iii) physiological variations and different contributions to the whole. Moreover, several technological and analytical problems and pitfalls that had to be surmounted during our studies focussed on the extensive qualitative and quantitative characterization of salivary peptidoma and mainly based on LC-MS analyses of intact naturally occurring peptides are here described. The hope is that the information provided might be helpful to other groups engaged on the analysis of saliva or other body fluids for clinical applications.

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“…As a result of drastic sample simplification, positional proteomics analysis provides insights in a variety of post-translational modification (PTM) processes and proteolytic processing that proteins may undergo at their N-terminal and C-terminal ends (5,6). Positional proteomics strategies rely on the ability to differentiate between the N-or C-terminal parts of a protein and the internal counterparts (7,8). Protocols for target enrichment of C-terminal peptides have only recently been introduced (9,10), mainly because of lower chemical reactivity of C-terminal carboxyl groups compared with N-terminal amino groups.…”

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confidence: 99%

Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion

Mommen

Waterbeemd

Meiring

et al. 2012

Molecular & Cellular Proteomics

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A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO 2 ) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO 2 , keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based

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Terminal proteomics: N‐ and C‐terminal analyses for high‐fidelity identification of proteins using MS

Cited by 46 publications

References 64 publications

Rapid sensitive analysis of cysteine rich peptide venom components

Rapid sensitive analysis of cysteine rich peptide venom components

Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us?

Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion

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