Multi-peptide nLC-PC-IDMS-SRM-based assay for the quantification of biomarkers in the chicken ovarian cancer model.
Methods
; 61(3): 323-30, 2013 Jun 15.
Article
en En
| MEDLINE
| ID: mdl-23603217
ABSTRACT
A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Neoplasias Ováricas
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Fragmentos de Péptidos
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Bioensayo
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Alfa-Macroglobulinas
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Prealbúmina
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Vitelogeninas
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Biomarcadores de Tumor
Tipo de estudio:
Guideline
/
Prognostic_studies
Límite:
Animals
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Female
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Humans
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos