Optimization of SELDI-TOF protein profiling for analysis of cervical mucous☆
Introduction
Cervical mucous, produced by secretory cells lining the cervix, consists of 2 major fractions, a viscoelastic gel phase consisting mainly of mucins, and an aqueous phase with lipids, proteins, enzymes and inorganic salts [1], [2]. It plays a vital role in the innate defense of the uterine cavity and also controls survival and migration of sperm. Cervical mucous is thought to be an ideal sample to screen for biomarkers of early detection of cervical cancer. As cervical mucous is produced in the microenvironment where cervical neoplasia arises, it is likely to include proteins produced by the lesion as well as by the host in response to the lesion. Also, the ability to use non-invasive collection methods is an added advantage for eventual clinical implementation.
While the cervical mucous proteome has not been examined in detail, several recent studies on cervico-vaginal fluid (CVF) that use a combination of proteomic tools, such as 1- or 2-dimensional gel electrophoresis and mass spectrometry (MS) [3], [4], [5] or liquid chromatography coupled to MS [4], [6], have been published. Several CVF proteins were noted to be of plasma origin, suggesting CVF could largely be plasma transduate. CVF is a combination of cervical mucous, vaginal fluid, oviductal fluid and endometrial fluid [7]. While cervical mucous and CVF will be expected to share similarities in their proteome, collection of cervical mucous using a sponge placed in the cervical os, could be expected to largely exclude vaginal fluid and result in differences as well.
One approach to biomarker discovery has been the use of high-throughput proteomic technologies such as the Surface-Enhanced Laser Desorption and Ionization Time of Flight mass spectrometry (SELDI-TOF MS) [8]. SELDI-TOF requires small sample amounts and little or no sample preparation because of its retentate chromatography feature. A majority of the studies conducted using SELDI-TOF have been for cancer biomarker analysis based on class comparison differences between subjects with tumors, including ovary, breast, lung among others, and those without tumors. The quest for these tumour markers has mainly been in serum and plasma but in some cases other body fluids such as urine, cerebrospinal fluid, sweat, tears and cellular secretions have been used [9], [10].
Biomarker analysis in these samples is challenging due to the large number of proteins that vary in concentration by log orders of magnitude in body fluids as well as the inherent individual to individual variation expected in the samples. Also, SELDI-TOF as a method has been criticized because of the lack of data reproducibility that arises mainly due to lack of standardization of pre-analytical and analytical phases [10], [11], [12], [13]. Hence, in this study several laboratory experimental parameters were evaluated, a combination of which would provide good quality and reproducible protein profiles of cervical mucous using SELDI-TOF MS. The restricted protein composition of cervical mucous is expected to improve the ability to obtain a fully representative proteome profile using this technology. The parameters selected here could in turn be applied in future studies to a larger sample set for biomarker discovery and validation for early detection of cervical cancer.
Section snippets
Sample collection
Cervical mucous samples were collected from women attending urban hospital colposcopy clinics who were enrolled as part of an ongoing study of cervical neoplasia [14]. Samples were collected at the time of colposcopy by absorption into two Weck-Cel® sponges (Xomed Surgical Products, Jacksonville, FL) placed, one at a time, into the cervical os [15]. Samples were stored at − 80 °C. This study was restricted to samples from women with no evidence of cervical neoplasia. Depending on the
Comparison of protein extraction procedures
Protein extraction buffers were evaluated based on the combination of ease of extraction, maximum protein content and maximum number of protein peaks with good intensity. Results from the total protein (Table 1) and SDS-PAGE (Fig. 2) analysis indicated that Denaturing buffer extracts had the highest protein content, followed closely by M-PER®. M-PER® and Cytobuster™ extracts gave the most complex profiles on NP-20, averaging 21 and 19 peaks between 2.5–30 kDa respectively (Fig. 3) and 5–7 peaks
Discussion
In order to reliably use SELDI-TOF protein profiles for biomarker discovery, it is important to optimize and standardize all steps of the process starting with experimental conditions to final data analysis. Cervical tissue as well as cervico-vaginal fluid has been previously analyzed by SELDI-TOF with respect to cervical disease and pregnancy [17], [18], [19], but to our knowledge, this is the first study to evaluate processing and assay conditions to improve SELDI-TOF protein spectra of
Acknowledgments
This work was supported in part by the National Cancer Institute's Early Detection Research Network (EDRN), Interagency Agreement Y1-CN-0101-01, Y1-CN-5005-01 and Oak Ridge Institute of Science and Education. We would like to thank Dr. Toni Whistler and Dr. Dominique Rollin, who first initiated SELDI studies of cervical mucous samples, for their comments and suggestions related to these experiments.
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