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Syphilis and the Host; multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

ABSTRACT: Whole cell lysates from hCMEC/d3 brain microvascular endothelial cells grown in medium and heavy SILAC media and exposed to Treponema pallidum or control media for 5 hours. Prior to Mass-spec analysis, each sample was fractionated into 12 fractions via high-pH reversed-phase liquid chromatography, then each fraction was analyzed by liquid chromatography tandem mass spectrometry. Exposures were repeated with 4 biological replicates, which we define as individual sample wells. In samples 1&2 medium SILAC cells were exposed to Treponema pallidum, and heavy SILAC cells were exposed to control media. In Samples 3&4 medium SILAC cells were exposed to control media, and heavy SILAC cells were exposed to Treponema pallidum. Cells were grown in D4-lysine (37.5 mg/liter) and 13C6-arginine (21.75 mg/liter) for medium isotope-labeled cells, or 13C615N2-lysine (38.5 mg/liter) and 13C615N4-arginine (22.25 mg/liter) for heavy isotope-labeled cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr. Caroline Cameron

PROVIDER: MSV000092275 | MassIVE | Mon Jun 26 16:37:00 BST 2023

SECONDARY ACCESSION(S): PXD043317

REPOSITORIES: MassIVE

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Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.

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Syphilis and the Host; multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

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