Dataset Information

ThinPrep Cervical Smear iTRAQ LC-MS/MS

ABSTRACT: The protein content from ThinPrep cervical smear specimens was purified with TCA precipitation. The protein pellets were dissolved and protein concentration was measured with the Bradford assay. 100 ug of protein for each sample were reduced with TCEP, cysteines were blocked with MMTS and finally digested overnight with trypsin. The resultant peptides were labeled with the iTRAQ 8plex reagents and after mixing were subjected to off-line high pH C18 fractionation. The peptide fractions were further analyzed with low pH C18 LC-Orbitrap Elite HCD MS/MS (MS resolution 120,000, top 10 precursors, isolation width 1.2 m/z, 100 min gradient method). Protein Identification was performed using Proteome Discoverer 1.3 software. SEQUEST node included prec. mass tolerance: 10 ppm, fragment mass tolerance 0.5 Da, iTRAQ8plex Nterm and K,Y static modification, Methylthio static modification, Phospho S variable modification, Oxidation M variable modification, Deamidation N,Q variable modification. All spectra were search against a Uniprot fasta file containing all reviewed Human entries and all reviewed and un-reviewed human papillomavirus entries. Peptide and PTM validation was performed with Percolator and PhosphoRS nodes respectively. Protein quantitation was performed with the Reporter Ions Quantifier node.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Theodoros Roumeliotis

PROVIDER: PXD000109 | Pride | 2013-05-09

REPOSITORIES: Pride

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Publications

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.

Papachristou Evaggelia K EK Roumeliotis Theodoros I TI Chrysagi Argyro A Trigoni Chrysanthi C Charvalos Ekatherina E Townsend Paul A PA Pavlakis Kitty K Garbis Spiros D SD

Journal of proteome research 20130422 5

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-Orbitra ...[more]

PMID: 23510160

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Project description:Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterised by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesise that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. Mass spectra were analyzed using Mascot (version 2.2, Matrix Science), executing a spectral search against a concatenated International Protein Index (IPI) human protein database (version 3.54 containing 39,925 entries) and a decoy database. Parameters included: trypsin enzyme specificity, SILAC double measurements of Lys6 and Arg8, 1 missed cleavage, minimum peptide length of 7 amino acids, minimum of 1 unique peptide, top 6 MS/MS peaks per 100 Da, peptide mass tolerance of 20 ppm for precursor ion and MS/MS tolerance of 0.5 Da. Oxidation of methionine and N-terminal protein acetylation for variable modifications and cysteine caramidomethylation for fixed modification. All entries were filtered using a false positive rate of 1% both at the peptide and protein levels, and false positives were removed. Quantification via normalised H/L ratios was based on minimum of 3 peptide ratio counts. Protein group entries with a normalised ratio significance B score of =< 0.05 or significance A score of =< 0.05 were retained for further analysis.

Dataset Information

ThinPrep Cervical Smear iTRAQ LC-MS/MS

Publications

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.

Similar Datasets

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