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

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.

Proteomics strategies to analyze HPV-transformed cells: relevance to cervical cancer

“…Recently, a study from Papachristou et al, allowed to detect six HPV-derived proteins, including the highrisk HPV16 type, in ThinPrep smear specimens from HPV positive and HPV negative females by a high precision Mud-PIT, iTRAQ labeling and high-resolution FT-Orbitrap MS. These data were also confirmed with PCR-hybridization molecular methods [35].…”

“…Shotgun analyses are commonly used on a standardized platform to compare proteome differences corresponding to disease phenotypes, molecular characteristics and responses to stimuli [34]. Recently, shotgun proteomics was applied to human thinprep cervical smear using isobaric tag for relative and absolute quantitation (iTRAQ) mass-tagging and 2D LC-FT-Orbitrap-MS [35].…”

“…Then, the identification of labeled peptides, by database searching, allows the identification of corresponding protein. iTRAQ technique was recently applied to human thin prep cervical smear HPV infected and non-providing a global whole new set of qualitative and quantitative data [35] SELDI is a variation of MALDI that uses a target modified to achieve biochemical affinity with the analyte compound. The analytical procedure begins with chromatographic separation where the sample is pre-fractionated by protein chip arrays with specific chromatographic features (cationic, anionic, hydrophobic, hydrophilic and ion metal chelating) [30].…”

Proteomic identification of potential biomarkers for cervical squamous cell carcinoma and human papillomavirus infection

“…17 The proteomics strategy based on isotopic labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents currently allows for the analysis of up to eight different samples simultaneously and quantitative detection of very low-abundance proteins, such as HPVderived proteins from cervical smears. [18][19][20] Previous proteomics studies have identified a number of biomarkers for early diagnosis or prognostication of cervical carcinoma and its precursors. 21 Because high-risk HPV types are frequently detected in most of the cases of cervical carcinoma and precancerous lesions, these biomarkers for cervical carcinoma are possibly associated with the HPV infection.…”

Biochemical and proteomic analysis of spliceosome factors interacting with intron-1 of human papillomavirus type-16

“…Proteomic and mass spectrometry technology might be an excellent tool to identify the nuclear factor which binds the HPV-16 In-1. In fact, this technology has been recently used to analyze human Thin Prep cervical smears from normal or cancer tissues, identifying inclusive protein from different HPV types [23].…”