Improving de Novo Sequencing of Peptides Using a Charged Tag and C-Terminal Digestion

Chen, Weibin; Lee, Peter J.; Shion, Henry; Ellor, Nicholas J; Gebler, John C.

doi:10.1021/ac061670b

Cited by 46 publications

(43 citation statements)

References 25 publications

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“…This charge derivatization produces better fragmentation and simpler spectra during collision activated dissociation experiments since an exclusive and often complete series of°N-terminal°fragment°ions°is°obtained° [34].°This derivatization procedure has also been applied successfully°to°tryptic°digests° [35].°In°the°case°of°MALDI experiments, where no additional proton is available that precludes the "mobile proton" model, this can be explained°by°charge-remote°fragmentation° [36,°37]. Very recently, coupling of TMPP derivatization with C-terminal digestion was shown to improve peptide de novo°sequencing°by°MALDI°MS° [38].…”

Section: Ecd Analysis Of Charge Derivatized (Tmpp) Peptidesmentioning

confidence: 99%

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

Chamot‐Rooke

Rest

Dalleu

et al. 2007

J. Am. Soc. Mass Spectrom.

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Electron capture dissociation (ECD) has become an alternative method to collision-activated dissociation (CAD) to avoid gas-phase cleavage of post-translational modifications carried by side chains from the peptide backbone. Nonetheless, as illustrated herein by the study of O-glycosylated and O-phosphorylated peptides, the extent of ECD fragmentations may be insufficient to cover the entire peptide sequence and to localize accurately these modifications. The present work demonstrates that the derivatization of peptides at their N-terminus by a phosphonium group improves dramatically and systematically the sequence coverage deduced from the ECD spectrum for both O-glycosylated and O-phosphorylated peptides compared with their native counterparts. The exclusive presence of N-terminal fragments (c-type ions) in the ECD spectra of doubly charged molecular cations simplifies peptide sequence interpretation. Thus, the combination of ECD and fixed charge derivatization appears as an efficient analytical tool for the extensive sequencing of peptides bearing labile groups. (J Am Soc Mass Spectrom 2007, 18, 1405-1413) © 2007 American Society for Mass Spectrometry T he field of proteomics usually aims at differential characterization of the complete pattern of protein expression, for instance between cells in a normal state and those in a pathological state. Protein identification is now routinely achieved on a large scale (tens or hundreds of proteins per day) by combining enzymatic digestion, mass spectrometric analysis of the resulting peptides, and subsequent database search. If few peptides or short peptide sequences are sufficient for protein identifications into databases, a complete determination of the primary structure is nevertheless useful to detect modifications that can be involved in the regulation of protein function. Indeed, the catalytic property, the conformation, the turnover, or the subcellular localization of proteins are largely under the control of various peptide sequence processings named posttranslational modifications. These modifications (PTM) involve the proteolytic maturation of the proteins and/or the transfer of chemical groups (phosphorylation, glycosylation, acetylation, sumoylation) on specific amino-acids (serine, threonine, or tyrosine for O-phosphorylations, serine or threonine for Oglycosylations, asparagine for N-glycosylations). The most obvious strategy for mapping the modifications along the peptide chain involves a comparison between the sequence obtained by mass spectrometric data and the one deduced from the open reading frame coded by the gene sequence. However, a drawback arises from the propensity of groups conjugated to the amino acid side chains to dissociate readily upon collisional activation. For instance, collisionally-activated dissociation (CAD) MS/MS spectra of protonated precursor ions of phosphorylated peptides exhibit intense ions originating from the elimination of metaphosphoric (HPO 3 , loss of 80 Da) or orthophosphoric acid (H 3 PO 4 , loss of 98 Da) [1]. I...

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Section: Ecd Analysis Of Charge Derivatized (Tmpp) Peptidesmentioning

confidence: 99%

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

Chamot‐Rooke

Rest

Dalleu

et al. 2007

J. Am. Soc. Mass Spectrom.

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“…For example, the reagent tris(trimethoxyphenyl) phosphonium acetic acid N-hydroxysuccinimidyl ester (TMPP-AcOSu) can introduce a positive phosphonium ion on the Ntermini of peptides [16,27,28]. It does not decompose to produce a mobile proton like TMAB.…”

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confidence: 99%

“…Several studies demonstrated that the phosphonium ion is absolutely fixed at the Nterminus, and N-terminal a n ions are produced [16][17][18][19]29]. Recent studies also indicate that the fragmentation efficiency of arginine-containing charge-tagged peptides is low [20,28,30]. In fact, barely any sequence information can be obtained.…”

mentioning

confidence: 99%

“…The result that arginine-containing charge-tagged peptides behave differently than other peptides is surprising, since if the charge is sequestered in the tag, all fragments should be generated by a charge-remote mechanism and the basicity of residues should not affect the peptide fragmentation. Nevertheless, because Cterminal arginine-containing peptides are commonly generated by trypsin digestion, different approaches have been investigated to facilitate the fragmentation of TMPP derivatized peptides [28,30]. Gebler et al [28] improved their fragmentation by removing the C-terminal basic residue.…”

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confidence: 99%

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Photodissociation of Charge Tagged Peptides

Parthasarathi

Raghavachari

et al. 2012

J. Am. Soc. Mass Spectrom.

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Tris(2,4,6-trimethoxyphenyl) phosphonium acetyl (TMPP-Ac) was previously introduced to improve the mass spectrometric sequence analysis of peptides by fixing a permanent charge at the N-termini. However, peptides containing arginine residues did not fragment efficiently after TMPP-Ac modification. In this work, we combine charge derivatization with photodissociation. The fragmentation of TMPP-derivatized peptides is greatly improved and a series of N-terminal fragments is generated with complete sequence information. Arginine has a special effect on the fragmentation of the TMPP tagged peptides when it is the N-terminal peptide residue. Theoretical and experimental results suggest that this is due to hydrogen transfer from the charged N-terminus to the hydrogen-deficient peptide sequence.

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“…Keough and colleagues found that derivatization helped them to get long un-interrupted ion ladders [19]. Derivatization methods that simplify MS/MS spectra and fix the precursor charge to one of the fragments have been described [20,21]. Although derivatization may aid in de novo sequencing, such methods may not be widely adapted since they incur additional cost and labor.…”

Section: Introductionmentioning

confidence: 99%

Algorithms for thede novosequencing of peptides from tandem mass spectra

Allmer¹

2011

Expert Review of Proteomics

107

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Proteomics is the study of the proteins, their time and location dependent expression profiles as well as their modifications and interactions. Mass spectrometry is useful to investigate many of the questions asked in proteomics. Typically database search methods are employed to identify proteins from complex mixtures. However, often databases are not available or despite their availability some sequences are not readily found therein. To overcome this problem de novo sequencing can be used to directly assign a peptide sequence to an MS/MS spectrum. Many algorithms have been proposed for de novo sequencing and a selection of them is detailed in this review. Although a standard accuracy measure has not been agreed upon in the field, relative algorithm performance is discussed. The current state of the de novo sequencing is assessed thereafter and finally, examples are used to construct possible future perspectives of the field. Running title:De novo sequencing of MS/MS spectra

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Improving de Novo Sequencing of Peptides Using a Charged Tag and C-Terminal Digestion

Cited by 46 publications

References 25 publications

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

Photodissociation of Charge Tagged Peptides

Algorithms for thede novosequencing of peptides from tandem mass spectra

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