title: Discovery and targeted proteomics on cutaneous biopsies infected by borrelia to investigate lyme disease.
abstract: Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, 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 gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy).
authors.affiliation: From the ‡Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS-Université de Strasbourg, 67087 Strasbourg, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Schnell
authors.affiliation: From the ‡Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS-Université de Strasbourg, 67087 Strasbourg, France.
authors.forename: Amandine
authors.initials: A
authors.lastname: Boeuf
authors.affiliation: From the ‡Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS-Université de Strasbourg, 67087 Strasbourg, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Westermann
authors.affiliation: ‖EA7290, Virulence bactérienne précoce, groupe Borréliose de Lyme, Facultés de Médecine et de Pharmacie, Université de Strasbourg, 67091 Strasbourg, France, and.
authors.forename: Benoît
authors.initials: B
authors.lastname: Jaulhac
authors.affiliation: **Faculté de Médecine, Université de Strasbourg et Clinique Dermatologique, Hôpitaux Universitaires, 67091 Strasbourg, France.
authors.forename: Dan
authors.initials: D
authors.lastname: Lipsker
authors.affiliation: From the ‡Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS-Université de Strasbourg, 67087 Strasbourg, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Carapito
authors.affiliation: ‖EA7290, Virulence bactérienne précoce, groupe Borréliose de Lyme, Facultés de Médecine et de Pharmacie, Université de Strasbourg, 67091 Strasbourg, France, and.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Boulanger
authors.affiliation: From the ‡Laboratoire de Spectrométrie de Masse BioOrganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS-Université de Strasbourg, 67087 Strasbourg, France, laurence.sabatier@unistra.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Ehret-Sabatier
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis*; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D001706:Biopsy; D025065:Borrelia burgdorferi / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid; D004586:Electrophoresis; D005408:Flagellin / Q000032:analysis*; D005782:Gels; D006801:Humans; D007553:Isotope Labeling; D008193:Lyme Disease / Q000175:diagnosis* / Q000382:microbiology; D051379:Mice; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012867:Skin / Q000382:microbiology / Q000473:pathology
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D005782:Gels; C079616:OspC protein; D010455:Peptides; D005408:Flagellin