Extensive research into the genomic and transcriptomic profiles of cancer has identified important biomarkers of prognosis. High-throughput profiling of the cancer proteome will give us further insight into factors that drive the malignant phenotype and underlie treatment response and resistance in individual ovarian cancer (OC) sub-types. The feasibility of reliable proteomics analysis of tissues has been enabled by using Pressure Cycling Technology (PCT) to digest small tissue samples, followed by SWATH-MS. Advantages of SWATH-MS include favourable sensitivity and reproducibility comparable to targeted proteomics, but without the need to optimise an assay prior to data collection.
Cancer tissues are complex specimens and some tumours, including OC, can be large. An important requirement for comparative proteomic analysis is that the effect of tissue composition and sub-sampling is understood. Therefore, the aims of this study are to i) establish a pre-analytical workflow for PCT-SWATH-MS analysis of OC and ii) investigate the inter- and intra-individual proteomic variability in multiple sub-samples from primary and matched metastatic disease.
Eleven high-grade serous OC cases were identified for this pilot study through the Gynaecology Oncology Biobank at Westmead Hospital. To assess tissue content, each specimen was cryo-sectioned and stained with haematoxylin and eosin (H&E). Two to five areas with varying cancer and stromal content were cored from frozen tissue (CryoXtract CXT350). A second H&E stained section was taken to confirm the cored areas. The cores were transected to give smaller segments (4-11 segments per core). The median wet weight of the samples was 2.5 mg (ranging from 0.4-6.3 mg). A total of 469 sub-samples from 21 matched primary and metastatic specimens were prepared for PCT-SWATH-MS.
A pre-analytical workflow for coring frozen ovarian tumours has been established and methods have been optimised for sample processing. A strategy was developed to reduce potential batch effects and samples analysed by SWATH-MS.