Comprehensive Library Generation for Identification and Quantification of Endometrial Cancer Protein Biomarkers in Cervico-Vaginal Fluid

Abstract

Endometrial cancer is the most common gynaecological malignancy in high-income countries and its incidence is rising. Early detection, aided by highly sensitive and specific biomarkers, has the potential to improve outcomes as treatment can be provided when it is most likely to effect a cure. Sequential window acquisition of all theoretical mass spectra (SWATH-MS), an accurate and reproducible platform for analysing biological samples, offers a technological advance for biomarker discovery due to its reproducibility, sensitivity and potential for data re-interrogation. SWATH-MS requires a spectral library in order to identify and quantify peptides from multiplexed mass spectrometry data. Here we present a bespoke spectral library of 154,206 transitions identifying 19,394 peptides and 2425 proteins in the cervico-vaginal fluid of postmenopausal women with, or at risk of, endometrial cancer. We have combined these data with a library of over 6000 proteins generated based on mass spectrometric analysis of two endometrial cancer cell lines. This unique resource enables the study of protein biomarkers for endometrial cancer detection in cervico-vaginal fluid. Data are available via ProteomeXchange with unique identifier PXD025925

HE4 as a Biomarker for Endometrial Cancer

“…The anatomical continuity of the uterine cavity with the lower genital tract has also led to interest in cervico-vaginal samples as a source of biomarkers for EC. Most of the literature around urogenital sampling for EC has involved cytological diagnosis, obtaining cytology samples from either the cervix or the uterine cavity directly, with mixed accuracy [ 114 ]. A recent study by O’Flynn et al reported a less invasive method of sampling using a Delphi screener, which does not require a full speculum examination, and reported pain scores similar to that of TVUS [ 112 ].…”

“…However, due to its role in immunity, the levels may be affected by the vaginal microbiome at the time of sampling [ 113 ]. There have been no studies investigating vaginal HE4 as a potential biomarker for EC, although work in this area is underway [ 114 ].…”

A Prostate Cancer Proteomics Database for SWATH-MS Based Protein Quantification

“…To increase the signal-to-noise ratio when building bespoke spectral libraries from clinical samples several strategies can be employed. First, one may be able to adopt a pooling strategy, grouping the samples in sub-pools to increase the probability to detect low abundance proteins or putative “sample-specific proteins” as our group has previously undertaken in other studies [ 24 ]. However, this current study has been performed in serum samples and not in proximal fluids.…”

“…For both analyses, the TOF-MS mass range was 400–1250 Da, and the product ion mass range was 100–1500 Da [ 46 ]. Additionally, 5 µL (~1 μg) of each reconstituted sample was loaded on a 10 cm fused silica AcclaimTM PepMapTM 5 μm 100 Nano-Trap Column (Thermo Fisher Scientific, Loughborough, UK) using a U3000 RSLC high-pressure nanoLC (Dionex, Cambereley, UK) as described by Njoku et al Eluting peptides were quantified using a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer operating in DDA mode [ 24 ].…”

“…This setting permits the incorporation of all y and b ion series especially y10 or y11, but only if they are particularly intense and observable. Previous investigations in our laboratory have demonstrated that these settings have no effect on quantification when a larger number of less intense ions are included [ 24 ]. To generate the in silico library, a list of proteins associated with PCa was collected from the Jensen lab’s “Disease database” [ 47 ], using a z-score cut-off of 5.0.…”