Analysis of Posttranslational Modifications of Proteins by Tandem Mass Spectrometry

Larsen, Martin R.; Trelle, Morten Beck; Thingholm, Tine E.; Jensen, Ole Mejlhede

doi:10.2144/000112201

Cited by 213 publications

(163 citation statements)

References 74 publications

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“…More significantly, in other cases, difficulties arise from the failure of peptide precursor ions to yield fragmentation informative of primary sequence. For example, the predominance of a single or few fragmentation pathways (as may be observed with post-translationally modified structures) may preclude the direct derivation of significant sequence information [4]. Furthermore, CAD (and other ion heating methods such as infra-red multiphoton dissociation) necessarily becomes less effective as the number of vibrational degrees of freedom in the precursor ion increases.…”

mentioning

confidence: 99%

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

Hart

Lau

Hao

et al. 2009

J. Am. Soc. Mass Spectrom.

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The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD-or ETD-based tandem mass spectrometry alone. (J Am Soc Mass Spectrom 2009, 20, 167-175)

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mentioning

confidence: 99%

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

Hart

Lau

Hao

et al. 2009

J. Am. Soc. Mass Spectrom.

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“…This technology presents important advantages. Apart from a high sensitivity, it can be applied to identify new posttranslational modifications and to determine the modification site [85]. Conversely, some drawbacks of this technique include the need for prior purification of the protein and a lack of compatibility between some existing MS-based methods and hydrophobic proteins.…”

Section: Mass Spectrometrymentioning

confidence: 99%

The Protein Lipidation and Its Analysis

Triola¹

2012

J Glycom Lipidom

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“…Mass spectrometry is the method of choice for the analysis of post-translational modifications both in the characterization of individual proteins and in large scale, systems biology experiments [14].…”

Section: Introductionmentioning

confidence: 99%

Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

Darula

Medzihradszky

2015

Anal. Chem.

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Iodoacetamide is perhaps the most widely used reagent for the alkylation of free sulfhydryls in proteomic experiments. Here we report that both incomplete derivatization of Cys side-chains and overalkylation of the peptides may lead to the misassignment of glycoforms when LC-MS/MS with electron-transfer dissociation (ETD) alone is used for the structural characterization of glycopeptides.Accurate mass measurements do not help, because the elemental compositions of the misidentified and correct modifications are identical. Incorporation of "higher-energy C-trap dissociation" (HCD), i.e. beam-type collision-induced dissociation data into the database searches with ETD data may prove decisive in most cases. However, the carbamidomethylation of Met residues leads to sulfonium ether formation, and the resulting fixed positive charge triggers a characteristic fragmentation, that eliminates the normal Y 1 fragment from the HCD spectra of N-linked glycopeptides, producing an abundant Y 1 -48 Da ion instead (the nominal mass diference is given relative to the unmodified amino acid sequence), that easily can be mistaken for the side-chain loss from Met sulfoxide. In such cases, good quality ETD data may indicate the discrepancy, and will also display abundant fragments due to CH 3 -S-CH 2 CONH 2 elimination from the charge-reduced precursor ions. Our observations also draw attention to the underreported interference of different unanticipated covalent modifications.

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Analysis of Posttranslational Modifications of Proteins by Tandem Mass Spectrometry

Cited by 213 publications

References 74 publications

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

The Protein Lipidation and Its Analysis

Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

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