The Shotgun Proteomic Study of the Human ThinPrep Cervical Smear Using iTRAQ Mass-Tagging and 2D LC-FT-Orbitrap-MS: The Detection of the Human Papillomavirus at the Protein Level
| Autor: | Argyro Chrysagi, Ekatherina Charvalos, Theodoros I. Roumeliotis, Kitty Pavlakis, Spiros D. Garbis, Evaggelia K. Papachristou, Chrysanthi Trigoni, Paul A. Townsend |
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| Rok vydání: | 2013 |
| Předmět: |
Proteomics
Proteome Collision-induced dissociation Molecular Sequence Data Uterine Cervical Neoplasms Shotgun Tandem mass spectrometry Mass spectrometry Sensitivity and Specificity Biochemistry Viral Proteins Tandem Mass Spectrometry Humans Amino Acid Sequence Human papillomavirus Vaginal Smears Human papillomavirus 16 Chromatography Chemistry Papillomavirus Infections Orbitrap ms Protein level Molecular Sequence Annotation General Chemistry Isotope Labeling Female |
| Zdroj: | Journal of Proteome Research. 12:2078-2089 |
| ISSN: | 1535-3907 1535-3893 |
| Popis: | The ThinPrep cervical smear is widely used in clinical practice for the cytological and molecular screening against abnormal cells and Human Papillomavirus (HPV) infection. Current advancements made to LC-MS proteomics include the use of stable isotope labeling for the in-depth analysis of proteins in complex clinical specimens. Such approaches have yet to be realized for ThinPrep clinical specimens. In this study, an LC-MS method based on isobaric (iTRAQ) labeling and high-resolution FT-Orbitrap mass spectrometry was used for the proteomic analysis of 23 human ThinPrep smear specimens. Tandem mass spectrometry analysis was performed with both nitrogen high collision dissociation (HCD MS/MS) and helium collision induced dissociation (CID MS/MS) peptide fragmentation modes. The analysis of three 8-plex sample sets yielded the identification of over 3200 unique proteins at FDR1%, of which over 2300 proteins were quantitatively profiled in at least one of the three experiments. The interindividual variability served to define the required sample size needed to identify significant protein expression differences. The degree of in-depth proteome coverage allowed the detection of 6 HPV-derived proteins including the high-risk HPV16 type in the specimens tested. The presence of the HPV strains of origin was also confirmed with PCR-hybridization molecular methods. This proof-of-principle study constitutes the first ever report on the nontargeted analysis of HPV proteins in human ThinPrep clinical specimens with high-resolution mass spectrometry. A further testament to the sensitivity and selectivity of the proposed study method was the confident detection of a significant number of phosphopeptides in these specimens. |
| Databáze: | OpenAIRE |
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