Basic abundance variation of S100 calcium-dependent family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.

<p>Basic abundance variation of S100 calcium-dependent family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.</p

Unbiased Proteomics Analysis Demonstrates Significant Variability in Mucosal Immune Factor Expression Depending on the Site and Method of Collection

<div><p>Female genital tract secretions are commonly sampled by lavage of the ectocervix and vaginal vault or via a sponge inserted into the endocervix for evaluating inflammation status and immune factors critical for HIV microbicide and vaccine studies. This study uses a proteomics approach to comprehensively compare the efficacy of these methods, which sample from different compartments of the female genital tract, for the collection of immune factors. Matching sponge and lavage samples were collected from 10 healthy women and were analyzed by tandem mass spectrometry. Data was analyzed by a combination of differential protein expression analysis, hierarchical clustering and pathway analysis. Of the 385 proteins identified, endocervical sponge samples collected nearly twice as many unique proteins as cervicovaginal lavage (111 vs. 61) with 55% of proteins common to both (213). Each method/site identified 73 unique proteins that have roles in host immunity according to their gene ontology. Sponge samples enriched for specific inflammation pathways including acute phase response proteins (p = 3.37×10⁻²⁴) and LXR/RXR immune activation pathways (p = 8.82×10⁻²²) while the role IL-17A in psoriasis pathway (p = 5.98×10⁻⁴) and the complement system pathway (p = 3.91×10⁻³) were enriched in lavage samples. Many host defense factors were differentially enriched (p<0.05) between sites including known/potential antimicrobial factors (n = 21), S100 proteins (n = 9), and immune regulatory factors such as serpins (n = 7). Immunoglobulins (n = 6) were collected at comparable levels in abundance in each site although 25% of those identified were unique to sponge samples. This study demonstrates significant differences in types and quantities of immune factors and inflammation pathways collected by each sampling technique. Therefore, clinical studies that measure mucosal immune activation or factors assessing HIV transmission should utilize both collection methods to obtain the greatest representation of immune factors secreted into the female genital tract.</p></div

Basic abundance variation of general antimicrobial factors collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.

<p>Basic abundance variation of general antimicrobial factors collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.</p

Protein pathways selectively enriched based on FGT compartment/collection method.

*<p>Denotes the number of protein factors identified in the proteomic dataset out of the total number of listed proteins involved in the pathway based on the Ingenuity software platform.</p

The biological functions of the proteins determined via hierarchical cluster analysis.

<p>Proteins associated with Branch 1 (i) and Branch 2 (ii) of the hierarchical cluster analysis according to their gene ontology determined via the functional annotation tool from DAVID Bioinformatics Resources.</p

Basic abundance variation of serpin family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.

<p>Basic abundance variation of serpin family members collected via Weck-Cel cervical sponge and cervicovaginal lavage as determined by mass spectrometry.</p

Detection of proteins associated with the CJIE4 prophage after growth on Mueller-Hinton agar in iTRAQ labelling experiments.

<p>Expression of proteins from <i>C. jejuni</i> isolate 00-2425 was assessed in previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095349#pone.0095349-Clark4" target="_blank">[10]</a>; -, not detected.</p

Comparison of sequence annotation for five CJIE4 prophages.

<p>Comparison of sequence annotation for five CJIE4 prophages.</p

Protein abundance patterns differentiate proteins identified and recovered by both collection methods.

<p>the Weck-Cel cervical sponge method (CER) and the cervicovaginal lavage method (CVL) (Proteins shown (121) were identified as differentially abundant by one-way ANOVA (p<0.05)). Clustering of proteins was generated by unsupervised centroid linkage hierarchical clustering using the Pearson correlation coefficient as the distance metric. Protein abundance levels are shown in colour, with red indicating overabundant proteins and green indicating underabundant proteins compared to the median of all samples for either collection method.</p

Comparison of putative phage repressor proteins associated with CJIE4 with lambda repressors.

<p>Comparison of putative phage repressor proteins associated with CJIE4 with lambda repressors.</p