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Discovery proteomics on cutaneous biopsies
Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge. The disease diagnosis relies mainly on clinical practice that can be coupled to serological or molecular diagnostics. However the relatively low sensitivity and specificity of the available Lyme diagnostics indicate the urgent need to identify additional highly antigenic borrelial proteins or to develop new strategies of diagnostic. As the skin constitutes a key interface where the pathogens can persist and multiply, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery Ge-LC-MS/MS approach on a murine model infected by B. burgdorferi sensu stricto. This dataset contains all the results concerning this approach.
Sample Processing Protocol
All the mouse skin biopsies were extracted using a Laemmli buffer. An equivalent of 50µg of proteins was loaded on a SDS-based gel. After protein migration, gel was cut in 25 bands and proteins were digested in-gel overnight. Peptides were then extracted and analysed by nano-LC-MS/MS on an ion trap (AmaZon, Bruker).
Data Processing Protocol
Mass data collected during nanoLC-MS/MS were processed, converted into â€œ.mgfâ€� files with DataAnalysis 4.0 (Bruker Daltonics) and interpreted using MASCOT 2.4.3 (Matrix Science, London, UK) and the OMSSA 2.1.7 (Open Mass Spectrometry Search Algorithm) algorithm run on the MSDA software suite (Mass Spectrometry Data Analysis). Searches were performed without any molecular weight, or isoelectric point restrictions against an in house generated protein database composed of all protein sequences of B. burgdorferi B31 and mouse (extracted from NCBInr and UniProtKB-SwissProt, respectively). Known contaminant proteins such as human keratins and trypsin, were added to the database and concatenated with reversed copies of all sequences (B. burgdorferi B31: August 16, 2012, 1758 entries; Mouse: April 19, 2013, 16722 entries). The database for B. burgdorferi B31 was used as the B. burgdorferi 297 strain has not been sequenced yet. Trypsin was selected as enzyme and for MS/MS data, a parent and fragment mass tolerance of 0.5 Da. A maximum of 2 missed cleavages was allowed and some modifications were taken into account: carbamidomethyl (C), acetyl N-terminus of protein, oxidation (M). The Mascot and OMSSA results were independently loaded into the Scaffold software (Proteome Software, Portland, OR) to validate peptide identifications. The target-decoy database search allowed us to control the false positive identification rate which was set to 1% with a minimum of one peptide per protein.
Schnell G, Boeuf A, Westermann B, Jaulhac B, Lipsker D, Carapito C, Boulanger N, Ehret-Sabatier L. Discovery and targeted proteomics on cutaneous biopsies infected by borrelia to investigate lyme disease. Mol Cell Proteomics. 2015 May;14(5):1254-64 PubMed: 25713121
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||35955||Discovery/shotgun proteomics of Borrelia infected mouse skin biopsies||1293||10108||4356||86837||10108||
|2||35942||Analyses of mouse skin biopsies infected by B. burgdorferi ss||23550||73785||59210||86837||73785||
|3||35954||Analyses of mouse skin biopsies infected by B. burgdorferi ss||2397||27210||10387||241025||27210||
|4||35941||Analyses of mouse skin biopsies nfected by B. burgdorferi ss||31092||194112||142764||241025||194112||
|5||35953||Discovery/shotgun proteomics of Borrelia infected mouse skin biopsies||1191||9308||3932||85562||9308||
|6||35944||Analyses of mouse skin biopsies infected by B. burgdorferi ss||23577||73813||58597||85562||73813||
|7||35943||Analyses of mouse skin biopsies infected by B. burgdorferi ss||1339||9585||4663||92715||9585||
|8||35952||Analyses of mouse skin biopsies infected by B.burgdorferi ss||24495||77773||64206||92715||77773||
|9||35951||Discovery/shotgun proteomics of Borrelia infected mouse skin biopsies||1422||9218||4677||90354||9218||
|10||35950||Analyses of mouse skin biopsies infected by B. burgdorferi ss||24414||77242||64210||90354||77242||