Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies

<p>Abstract</p> <p>Background</p> <p>Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value.</p> <p>Aim</p> <p>To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3.</p> <p>Methods</p> <p>Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications.</p> <p>Results</p> <p>The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification.</p> <p>Conclusions</p> <p>114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.</p

An evaluation of corallophelia pertusamucus as an analytical matrix for environmental monitoring: A preliminary proteomic study

For the environmental monitoring of coral, mucus appears to be an appropriate biological matrix due to its array of functions in coral biology and the non-intrusive manner in which it can be collected. The aim of the present study was to evaluate the feasibility of using mucus of the stony coral Lophelia pertusa (L. pertusa) as an analytical matrix for discovery of biomarkers used for environmental monitoring. More specifically, to assess whether a mass-spectrometry-based proteomic approach can be applied to characterize the protein composition of coral mucus and changes related to petroleum discharges at the seafloor. Surface-enhanced laser desorption/ionization–time of flight mass spectrometry (SELDI-TOF MS) screening analyses of orange and white L. pertusa showed that the mucosal protein composition varies significantly with color phenotype, a pattern not reported prior to this study. Hence, to reduce variability from phenotype difference, L. pertusa white individuals only were selected to characterize in more detail the basal protein composition in mucus using liquid chromatography, mass spectrometry, mass spectrometry (LC-MS/MS). In total, 297 proteins were identified in L. pertusa mucus of unexposed coral individuals. Individuals exposed to drill cuttings in the range 2 to 12 mg/L showed modifications in coral mucus protein composition compared to unexposed corals. Although the results were somewhat inconsistent between individuals and require further validation in both the lab and the field, this study demonstrated preliminary encouraging results for discovery of protein markers in coral mucus that might provide more comprehensive insight into potential consequences attributed to anthropogenic stressors and may be used in future monitoring of coral health