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
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
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 FDR < 1%, 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.
2078-2089
Papachristou, Evaggelia K.
32689d8c-a4f1-447b-9983-c82e4790f234
Roumeliotis, Theodoros I.
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Chrysagi, Argyro
2624698b-9b23-4de3-93df-06cf94c04484
Trigoni, Chrysanthi
58c45050-a3f9-4853-903d-d37746f95241
Charvalos, Ekatherina
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Townsend, Paul A.
a2680443-664e-46d0-b4dd-97456ba810db
Pavlakis, Kitty
cb774e11-a435-4584-9d43-2cd9690bc606
Garbis, Spiros
7067fd19-50c9-4d42-9611-f370289470bd
22 April 2013
Papachristou, Evaggelia K.
32689d8c-a4f1-447b-9983-c82e4790f234
Roumeliotis, Theodoros I.
23f78732-120b-407c-97bb-c73022305810
Chrysagi, Argyro
2624698b-9b23-4de3-93df-06cf94c04484
Trigoni, Chrysanthi
58c45050-a3f9-4853-903d-d37746f95241
Charvalos, Ekatherina
c8362032-5b9f-488a-b132-b64b249f87e1
Townsend, Paul A.
a2680443-664e-46d0-b4dd-97456ba810db
Pavlakis, Kitty
cb774e11-a435-4584-9d43-2cd9690bc606
Garbis, Spiros
7067fd19-50c9-4d42-9611-f370289470bd
Papachristou, Evaggelia K., Roumeliotis, Theodoros I., Chrysagi, Argyro, Trigoni, Chrysanthi, Charvalos, Ekatherina, Townsend, Paul A., Pavlakis, Kitty and Garbis, Spiros
(2013)
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.
Journal of Proteome Research, 12 (5), .
(doi:10.1021/pr301067r).
(PMID:23510160)
Abstract
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 FDR < 1%, 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.
Text
The Shotgun Proteomic Study of the Human ThinPrep Cervical Smear Using iTRAQ Mass-Tagging and 2D LC-FT-Orbitrap-MS.pdf
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e-pub ahead of print date: 19 March 2013
Published date: 22 April 2013
Organisations:
Cancer Sciences
Identifiers
Local EPrints ID: 395405
URI: http://eprints.soton.ac.uk/id/eprint/395405
ISSN: 1535-3893
PURE UUID: 96824116-66e8-4890-90a2-550058fda92a
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Date deposited: 15 Jul 2016 14:28
Last modified: 15 Mar 2024 00:39
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Contributors
Author:
Evaggelia K. Papachristou
Author:
Theodoros I. Roumeliotis
Author:
Argyro Chrysagi
Author:
Chrysanthi Trigoni
Author:
Ekatherina Charvalos
Author:
Paul A. Townsend
Author:
Kitty Pavlakis
Author:
Spiros Garbis
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