Neutron-encoded signatures enable product ion annotation from tandem mass spectra.
Mol Cell Proteomics
; 12(12): 3812-23, 2013 Dec.
Article
en En
| MEDLINE
| ID: mdl-24043425
We report the use of neutron-encoded (NeuCode) stable isotope labeling of amino acids in cell culture for the purpose of C-terminal product ion annotation. Two NeuCode labeling isotopologues of lysine, (13)C6(15)N2 and (2)H8, which differ by 36 mDa, were metabolically embedded in a sample proteome, and the resultant labeled proteins were combined, digested, and analyzed via liquid chromatography and mass spectrometry. With MS/MS scan resolving powers of ~50,000 or higher, product ions containing the C terminus (i.e. lysine) appear as a doublet spaced by exactly 36 mDa, whereas N-terminal fragments exist as a single m/z peak. Through theory and experiment, we demonstrate that over 90% of all y-type product ions have detectable doublets. We report on an algorithm that can extract these neutron signatures with high sensitivity and specificity. In other words, of 15,503 y-type product ion peaks, the y-type ion identification algorithm correctly identified 14,552 (93.2%) based on detection of the NeuCode doublet; 6.8% were misclassified (i.e. other ion types that were assigned as y-type products). Searching NeuCode labeled yeast with PepNovo(+) resulted in a 34% increase in correct de novo identifications relative to searching through MS/MS only. We use this tool to simplify spectra prior to database searching, to sort unmatched tandem mass spectra for spectral richness, for correlation of co-fragmented ions to their parent precursor, and for de novo sequence identification.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Algoritmos
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Proteoma
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Proteínas de Saccharomyces cerevisiae
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Anotación de Secuencia Molecular
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Marcaje Isotópico
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Neutrones
Idioma:
En
Revista:
Mol Cell Proteomics
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2013
Tipo del documento:
Article
Pais de publicación:
Estados Unidos