Abstract
BackgroundCervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C4(RP)-LC protein separation) followed by C18(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.ResultsIn total, we were able to identify 339 proteins in human CVF of which 151 proteins were not identified in any other proteomics study on human CVF so far. Those included antimicrobial peptides, such as human beta-defensin 2 and cathelicidin, which were known to be present in CVF, and endometrial proteins such as glycodelin and ribonucleoprotein A. Comparison of our results with previously published data led to the identification of a common protein set of 136 proteins. This overlapping protein set shows increased fractions of immunological and extracellular proteins, confirming the extracellular immunological role of CVF.ConclusionWe demonstrated here that CVF colposcopy samples can be used in proteomics experiments and hence are applicable for biomarker discovery experiments. The delineation of an overlapping set of proteins that is identified in most proteomics studies on CVF may help in the description of a reference proteome when performing proteomics studies on human CVF.
Highlights
Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, little is known about the contribution of CVF proteins to the immunity of the lower female genital tract
We demonstrated here that CVF colposcopy samples can be used in proteomics experiments and are applicable for biomarker discovery experiments
Since it is well described that usage of different fractionation techniques enhances the dynamic range of the experimental setup and increases proteome coverage[45], we analyzed the effect of the incorporation of an ultrafiltration step, prior to the C4(reversed-phase (RP))-liquid chromatography (LC) fractionation on the protein level, on the number of protein identifications obtained
Summary
Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. The innate immunity of the mucosa of the female lower genital tract has antimicrobial proteins/peptides (AMPs) (e.g. defensins, lactoferrin, cathelicidin, lysozyme, SLPI, etc.) as predominant effector molecules, which are present in cervicovaginal mucosal and glandular excretions (reviewed in[1]). Structurally diverse, they are often small (< 100 amino acids), cationic and amphipathic molecules[12]. Aside from these activities, they have effects on the host's immunity[12] and on the target cells of viruses and bacteria [1820]
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