A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Abstract

BackgroundThe proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.ResultsThe average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.ConclusionsResidual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.

Candidate biomarkers in the cervical vaginal fluid for the (self-)diagnosis of cervical precancer

“…Using the emerging proteomics technologies that have become increasingly sensitive, our group and others groups have conducted several studies on the identification of the cervical vaginal proteome [ 48 – 65 ]. Functional classification of the CVF proteome demonstrates a significant diversity of biological roles, of which “protein metabolism and modification” and “immunity and defense” are the largest GO categories (17 and 10%, respectively).…”

Evaluating the potential of residual Pap test fluid as a resource for the metaproteomic analysis of the cervical-vaginal microbiome

“…It is not clear why the number of peptides assigned varied so greatly between NPap933 and the samples from the four other individuals. In our previous analysis of human proteins present in residual Pap test fixative, we identified between 317 and 500 proteins from these same Pap samples 21 . While it is tempting to think that this might reflect a difference in the relative abundance of bacterial colonization of those individuals, the low number of microbial peptides recovered from individual samples NPap94, NPap134, and NPap137 could also be due to limitations in the way those samples were collected and processed.…”

“…We previously demonstrated the feasibility of using the residual fixative from discarded Pap tests for the identification of human cervical-vaginal proteins by mass spectrometry 21 . Screening for cervical cancer by Pap tests has been performed routinely for over 50 years 22 and over 30 million are performed annually in the U.S 23 .…”

“…The liquid fixative solution in which the cells are collected for Pap tests is discarded after examination of the cells. Using this unique sample source, we identified over 400 proteins from the Human Uniprot database, and defined a “Normal Pap test Core Proteome” consisting of 153 proteins 21 . To evaluate this approach for the identification of taxonomic and functional information on the vaginal microbiome, these same raw data were analyzed using a two-step method for peptide sequence matching and protein identification for metaproteomic analysis of microbial peptides present in the residual Pap test fixative from women over age 50.…”

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

“…Since Papanicolaou (Pap) test was approved by the US Food and Drug Administration (FDA) in 1996, a vast majority of cervical cancer screening has used liquid-based Pap test 194 , 195 . Boylan et al examined the proteins present in residual Pap test fixative samples from females with normal cervical cytology by 2-D-MS/MS and created a “Normal Pap test Core Proteome” 196 . More recently, the same group used iTRAQ to quantify the proteins in Pap test samples from patients with ovarian cancer compared with healthy controls or patients with benign gynecological disease 197 .…”