Proteomics

Dataset Information

119

Unique insights in cervicovaginal Lactobacillus iners and L. crispatus proteome changes associated with microbiome dysbiosis


ABSTRACT: Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Lactobacillus iners   Homo sapiens   Lactobacillus crispatus  

TISSUE(S): Cervicovaginal Fluid

DISEASE(S): Not Available

SUBMITTER: Stuart Armstrong   

PROVIDER: PXD003176 | Pride | 2016-03-15

REPOSITORIES: Pride

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Publications

Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis.

Borgdorff Hanneke H   Armstrong Stuart D SD   Tytgat Hanne L P HL   Xia Dong D   Ndayisaba Gilles F GF   Wastling Jonathan M JM   van de Wijgert Janneke H H M JH  

PloS one 20160310 3


BACKGROUND:A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition. METHODS:The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovag  ...[more]

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