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• W2099950291 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). 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). Lyme borreliosis is an arthropod-borne disease transmitted by hard ticks (Ixodes spp.). The causative agents are bacteria belonging to the Borrelia burgdorferi sensu lato group. In the United States, more than 30,000 cases have been reported to the Centers for Disease Control and Prevention in 2012. There, the unique pathogenic species of Borrelia is B. burgdorferi sensu stricto (s.s.). In Europe, between 65,000 and 85,000 cases are reported depending on the epidemiological study (1Hubálek Z. Epidemiology of lyme borreliosis.Curr. Probl. Dermatol. 2009; 37: 31-50Crossref PubMed Scopus (172) Google Scholar, 2Lindgren E. Jaensson T.G.T. Lyme Borreliosis in Europe: Influences of Climate and Climate Change, Epidemiology, Ecology and Adaptation Measures.World Health Organization Europe. 2006; Google Scholar), and the three most prevalent pathogenic species of Borrelia are Borrelia afzelii, Borrelia garinii, and B. burgdorferi s.s. The disease in both Europe and the United States is first characterized in most patients by an inflammatory skin lesion, erythema migrans (EM), which is the most frequent manifestation of the disease. Dissemination to other sites occurs secondarily and can involve among others articulation, nervous system, heart, and skin at other sites (3Stanek G. Wormser G.P. Gray J. Strle F. Lyme borreliosis.Lancet. 2012; 379: 461-473Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 4Radolf J.D. Caimano M.J. Stevenson B. Hu L.T. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.Nat. Rev. Microbiol. 2012; 10: 87-99Crossref PubMed Scopus (492) Google Scholar). The diagnosis can be a real challenge because of the proteiform clinical manifestations. When an EM is present, which is the case for 80% of patients (3Stanek G. Wormser G.P. Gray J. Strle F. Lyme borreliosis.Lancet. 2012; 379: 461-473Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), early diagnosis is facilitated. However, EM presentation can be clinically atypical, making the recognition of this manifestation of Lyme borreliosis difficult (5Strle F. Stanek G. Clinical manifestations and diagnosis of lyme borreliosis.Curr. Probl. Dermatol. 2009; 37: 51-110Crossref PubMed Scopus (129) Google Scholar). Later on, when Borrelia has disseminated to the target organs, biological diagnosis is based either on the direct detection of the pathogen in different patient body fluids and biopsies by means of culture and/or PCR or on the indirect demonstration of presence of Borrelia by detection of anti-pathogen-directed IgM and IgG antibodies (enzyme-linked immunosorbent assay (ELISA) and Western blot) (6Aguero-Rosenfeld M.E. Wang G. Schwartz I. Wormser G.P. Diagnosis of lyme borreliosis.Clin. Microbiol. Rev. 2005; 18: 484-509Crossref PubMed Scopus (548) Google Scholar). Concerning the direct detection of Borrelia, culture of the bacteria has allowed the spirochete isolation since the 80s in different specific Barbour-Stoenner-Kelly-based media by using skin biopsies or biological fluids such as blood or cerebrospinal fluid (7Benach J.L. Bosler E.M. Hanrahan J.P. Coleman J.L. Habicht G.S. Bast T.F. Cameron D.J. Ziegler J.L. Barbour A.G. Burgdorfer W. Edelman R. Kaslow R.A. Spirochetes isolated from the blood of two patients with Lyme disease.N. Engl. J. Med. 1983; 308: 740-742Crossref PubMed Scopus (571) Google Scholar, 8Asbrink E. Hovmark A. Successful cultivation of spirochetes from skin lesions of patients with erythema chronicum migrans Afzelius and acrodermatitis chronica atrophicans.Acta Pathol. Microbiol. Immunol. Scand. B. 1985; 93: 161-163PubMed Google Scholar). However, Borrelia culture is not routinely used as a diagnostic test because the bacterial growth takes several weeks and does not yield timely results. Indeed, it requires the use of the specific and expensive Barbour-Stoenner-Kelly medium and a dark field microscope to detect, frequently after at least 2 weeks of incubation, the presence of Borrelia in tissues or biological fluids. When performed from patients with EM, only 40–80% of the cultures are positive (6Aguero-Rosenfeld M.E. Wang G. Schwartz I. Wormser G.P. Diagnosis of lyme borreliosis.Clin. Microbiol. Rev. 2005; 18: 484-509Crossref PubMed Scopus (548) Google Scholar). In addition, the success of culture varies greatly according to the Borrelia species. PCR is quicker and generally more sensitive than culture with a range of 36–88%, although the success of bacterial detection varies with the gene selected for the assay (6Aguero-Rosenfeld M.E. Wang G. Schwartz I. Wormser G.P. Diagnosis of lyme borreliosis.Clin. Microbiol. Rev. 2005; 18: 484-509Crossref PubMed Scopus (548) Google Scholar). PCR is efficient for Borrelia detection in synovial liquid (60–85% of the cases) in the case of arthritis (9Nocton J.J. Dressler F. Rutledge B.J. Rys P.N. Persing D.H. Steere A.C. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.N. Engl. J. Med. 1994; 330: 229-234Crossref PubMed Scopus (478) Google Scholar, 10Jaulhac B. Chary-Valckenaere I. Sibilia J. Javier R.M. Piémont Y. Kuntz J.L. Monteil H. Pourel J. Detection of Borrelia burgdorferi by DNA amplification in synovial tissue samples from patients with Lyme arthritis.Arthritis Rheum. 1996; 39: 736-745Crossref PubMed Scopus (110) Google Scholar) but less sensitive in cases of neuroborreliosis in cerebrospinal fluid (<20–40% of the cases) (9Nocton J.J. Dressler F. Rutledge B.J. Rys P.N. Persing D.H. Steere A.C. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.N. Engl. J. Med. 1994; 330: 229-234Crossref PubMed Scopus (478) Google Scholar, 11Wilske B. Fingerle V. Schulte-Spechtel U. Microbiological and serological diagnosis of Lyme borreliosis.FEMS Immunol. Med. Microbiol. 2007; 49: 13-21Crossref PubMed Scopus (207) Google Scholar). Moreover, PCR detects DNA and not proteins and therefore prevents the detection of active infection. As far as the skin biopsies are concerned, the sensitivity of detection is variable in cases of EM or acrodermatitis chronica atrophicans (12Nowakowski J. Schwartz I. Liveris D. Wang G. Aguero-Rosenfeld M.E. Girao G. McKenna D. Nadelman R.B. Cavaliere L.F. Wormser G.P. Lyme Disease Study Group Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a comparison of different techniques.Clin. Infect. Dis. 2001; 33: 2023-2027Crossref PubMed Scopus (116) Google Scholar). Conversely, indirect detection using serological tests is not adapted to the early diagnosis as it relies on antibodies only detectable after at least 4–6 weeks after the infectious tick bite. These tests also suffer from lack of specificity (13Marques A.R. Lyme disease: a review.Curr. Allergy Asthma Rep. 2010; 10: 13-20Crossref PubMed Scopus (62) Google Scholar). New diagnostic approaches are therefore required. Selected reaction monitoring (SRM) has been recognized as an efficient mass spectrometry-based technique for the biomarker verification and validation in several biological fluids (blood, plasma, and urine) (14Kuhn E. Whiteaker J.R. Mani D.R. Jackson A.M. Zhao L. Pope M.E. Smith D. Rivera K.D. Anderson N.L. Skates S.J. Pearson T.W. Paulovich A.G. Carr S.A. Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.Mol. Cell. Proteomics. 2012; 11Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility.Nat. Biotechnol. 2006; 24: 971-983Crossref PubMed Scopus (1369) Google Scholar, 16HÜttenhain R. Soste M. Selevsek N. Röst H. Sethi A. Carapito C. Farrah T. Deutsch E.W. Kusebauch U. Moritz R.L. Niméus-Malmström E. Rinner O. Aebersold R. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.Sci. Transl. Med. 2012; 4: 142ra94Crossref PubMed Scopus (209) Google Scholar, 17Percy A.J. Chambers A.G. Yang J. Borchers C.H. Multiplexed MRM-based quantitation of candidate cancer biomarker proteins in undepleted and non-enriched human plasma.Proteomics. 2013; 13: 2202-2215Crossref PubMed Scopus (60) Google Scholar, 18Kennedy J.J. Abbatiello S.E. Kim K. Yan P. Whiteaker J.R. Lin C. Kim J.S. Zhang Y. Wang X. Ivey R.G. Zhao L. Min H. Lee Y. Yu M.-H. Yang E.G. Lee C. Wang P. Rodriguez H. Kim Y. Carr S.A. Paulovich A.G. Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.Nat. Methods. 2014; 11: 149-155Crossref PubMed Scopus (145) Google Scholar). The demonstrated specificity, selectivity, and high sensitivity (low attomole range) of the technique (19Picotti P. Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions.Nat. Methods. 2012; 9: 555-566Crossref PubMed Scopus (996) Google Scholar) makes it promising for the development of an SRM-based method for early diagnosis of Lyme disease. To our knowledge, this strategy has only rarely been used on skin tissue (20Williamson J.C. Scheipers P. Schwämmle V. Zibert J.R. Beck H.C. Jensen O.N. A proteomics approach to the identification of biomarkers for psoriasis utilising keratome biopsy.J. Proteomics. 2013; 94: 176-185Crossref PubMed Scopus (30) Google Scholar). It would allow the direct and rapid detection of Borrelia proteins in the skin, demonstrating the presence of an active infection very early after the tick transmission. In the present study, we set up a workflow to develop a robust and sensitive SRM assay to detect Borrelia in human skin samples (Fig. 1). First, we looked for Borrelia proteins in infected mouse skin samples by using a classical shotgun/discovery strategy. This experiment afforded a list of bacterial proteins that are expressed in vivo in the skin of an infected mammalian host. Then, we selected protein targets and optimized a Ge-LC-SRM assay to specifically detect and quantify these proteins in mouse skin samples. We demonstrated the transferability of the SRM assay for the detection of the targeted proteins in human skin samples naturally infected with Borrelia. Finally, we improved the experimental protocol to avoid gel prefractionation. Modified porcine trypsin was obtained from Promega (Madison, WI). High quality and crude isotopically labeled standard peptides with C-terminal 15N- and 13C-labeled arginine and lysine residues (HeavyPeptide AQUATM Ultimate at 5 pmol/μl ± 5% and PEPotecsTM) were synthesized by Thermo Fisher Scientific (Bremen, Germany). C18 Sep-Pak cartridges (Sep-Pak Vac, 1 ml, 50 mg, tC18) were obtained from Waters (Milford, MA), and all other reagents and chemicals were purchased from Sigma-Aldrich. All buffers were prepared with ultrapure water. Mice were infected intradermally with 103 spirochetes of B. burgdorferi sensu stricto (strain 297) in 0.1 ml of Barbour-Stoenner-Kelly culture medium in the dorsal thoracic area. At two time points after the inoculation (days 5 and 7) mice were killed by isoflurane. An approximately 1-cm area of mouse skin was collected at the inoculation site. For the reproducibility study, mouse skin samples were cut in three equal parts around the inoculation point. Samples were stored at −80 °C until analysis. For human samples, 4-mm skin biopsies were taken after informed consent and local anesthesia from six patients with EM lesions (H1–H6) after a tick bite in France (ClinicalTrials.gov Identifier NCT00576082). Half of each biopsy was used for both culture and PCR, and half of the biopsy was immediately frozen and kept at −80 °C until use. These six biopsies were all positive by both culture and PCR, and the causative species were identified by a species-specific real time PCR assay (21Hidri N. Barraud O. de Martino S. Garnier F. Paraf F. Martin C. Sekkal S. Laskar M. Jaulhac B. Ploy M.-C. Lyme endocarditis.Clin. Microbiol. Infect. 2012; 18: E531-E532Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Biopsies H1, H2, H3, and H5 were taken from solitary EM lesions and were infected with B. afzelii, and biopsy H6 was infected with B. garinii. The causative species in biopsy H4, taken from a patient with multiple EM, was B. afzelii. For mouse and human skin biopsies, 4-mg samples were manually extracted by 200 μl of Laemmli sample buffer in a 0.1-ml Potter tissue grinder, Wheaton, Millville, NJ. After 5 min of sonication and 10 min of centrifugation (14,000 × g at 4 °C), the protein content of the supernatant was determined by using a detergent-compatible assay (Bio-Rad). Proteins (50 μg) were subjected to 12% SDS-PAGE and stained overnight with colloidal Coomassie Brilliant Blue (22Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1594) Google Scholar). 25 gel bands of 2 mm were excised manually. In-gel digestion was carried out as described previously (23Villiers C. Chevallet M. Diemer H. Couderc R. Freitas H. Van Dorsselaer A. Marche P.N. Rabilloud T. From secretome analysis to immunology: chitosan induces major alterations in the activation of dendritic cells via a TLR4-dependent mechanism.Mol. Cell. Proteomics. 2009; 8: 1252-1264Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and the tryptic peptides were extracted (60% ACN, 0.1% HCO2H) prior to mass spectrometry analyses. Six different extraction protocols compatible with liquid digestion were tested: (i) 1% RapiGestTM SF (Waters) in 50 mm NH4HCO3 buffer, (ii) 1% deoxycholate (DOC) in 25 mm NH4HCO3 buffer, (iii) 8 m urea in 100 mm NH4HCO3 buffer, (iv) 1% octyl β-d-glucopyranoside (octyl glucoside) in 10 mm NaCl, 10 mm NH4HCO3 buffer, (v) 2% SDS in 25 mm NH4CO3 buffer, and (vi) filter-aided sample preparation as described by Wiśniewski et al. (24Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5097) Google Scholar). The mouse skin biopsies were extracted in 200 μl of buffer in a 0.1-ml Potter tissue grinder. After 5 min of sonication and 10 min of centrifugation (14,000 × g at 4 °C), the protein content of the supernatant was determined by using a detergent-compatible assay. The filter-aided sample preparation protocol (vi) was then performed as described (24Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5097) Google Scholar) starting from 100 μg of proteins. For the other protocols (i–v), 100 μg of each extract was reduced for 1 h at 60 °C (except 37 °C for the urea protocol (iii)) by adding dithiothreitol to a final concentration of 10 mm. Alkylation was performed by adding iodoacetamide to a final concentration of 40 mm at room temperature. To carry out the digestion in an optimal way, the sample was diluted to 1 m urea (i) and 0.07% SDS (vi). For the octyl glucoside protocol (iv), proteins were precipitated by addition of ice-cold acetone overnight and solubilized in 0.1 m NH4HCO3 before digestion. An overnight digestion was performed by adding trypsin in a 1:50 enzyme to protein ratio. After digestion, trifluoroacetic acid was added to a final concentration of 0.5% (v/v) for protocols i, ii, iii, and iv. For protocol vi, 4 m KCl was added just after digestion to precipitate the SDS. A centrifugation step was then necessary to eliminate the by-products of RapiGest SF (i), the precipitated DOC (ii), and SDS (vi). All samples were desalted on Sep-Pak C18 cartridges and recovered in 100 μl of 0.1% HCO2H. The peptide content was determined by a detergent-compatible assay. One microliter of a mixture of heavy labeled peptides was finally added to a volume of sample solution containing 1 μg of peptides prior to SRM analyses. The efficiency of the different protocols was evaluated by calculating the extraction yield and the sample recovery. The extraction yield corresponds to the protein content obtained after the extraction step divided by the weight of the biopsy. The sample recovery is the peptide content after Sep-Pak desalting divided by the initial protein content and multiplied by 100. The RapiGest protocol was applied to one human skin biopsy (H1). The extraction, reduction, alkylation, and digestion steps were performed as for mouse skin biopsies. Peptides were analyzed on a nano-LC-Chip/Cube (Agilent Technologies, Palo Alto, CA) hyphenated to an amaZon ion trap (Bruker Daltonics, Bremen, Germany). The chip contained a Zorbax 300SB-C18 column (150 mm × 75 μm, 5 μm) and a Zorbax 300SB-C18 enrichment column (40 nl, 5 μm). The solvent system consisted of 2% ACN, 0.1% HCO2H in water (solvent A) and 2% water, 0.1% HCO2H in ACN (solvent B). Trapping was done at a flow rate set to 3.75 μl/min with solvent A. Elution was performed at a flow rate of 300 nl/min with a 8–40% linear gradient (solvent B) in 30 min followed by a 4-min stage at 70% solvent B before reconditioning the column at 8% solvent B. The MS spectra were acquired in the positive ion mode on the mass range 250–1500 m/z using the standard enhanced resolution mode at a scan rate of 8100 m/z/s. The eight most abundant peptides were selected on each MS spectrum for further isolation and fragmentation with a preference for doubly charged ions (absolute threshold of 100,000). Fragmentation was performed using argon as the collision gas. Ions were excluded after the acquisition of one MS/MS spectrum, and the exclusion was released after 0.60 min. MS/MS spectra were acquired on the mass range 100–2000 m/z. The complete system was fully controlled by HyStar 3.2 (Bruker Daltonics) software. Mass data collected during nano-LC-MS/MS were processed, converted into “.mgf” files with DataAnalysis 4.0 (Bruker Daltonics), and interpreted using Mascot 2.4.3 (Matrix Science, London, UK) and Open Mass Spectrometry Search Algorithm (OMSSA) 2.1.7 (25Geer L.Y. Markey S.P. Kowalak J.A. Wagner L. Xu M. Maynard D.M. Yang X. Shi W. Bryant S.H. Open mass spectrometry search algorithm.J. Proteome Res. 2004; 3: 958-964Crossref PubMed Scopus (1166) Google Scholar) algorithm run on the Mass Spectrometry Data Analysis (MSDA) software suite (26Carapito C. Burel A. Guterl P. Walter A. Varrier F. Bertile F. Van Dorsselaer A. MSDA, a proteomics software suite for in-depth mass spectrometry data analysis using grid computing.Proteomics. 2014; 14: 1014-1019Crossref PubMed Scopus (44) Google Scholar). 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-Swiss-Prot, 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, 16,722 entries). The database for B. burgdorferi B31 was used because 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 was used. A maximum of two missed cleavages was allowed, and some modifications were taken into account: carbamidomethyl (Cys), acetyl N terminus of protein, and oxidation (Met). The Mascot and OMSSA results were independently loaded into 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. Borrelia protein identifications in mouse skin biopsies analyzed are listed in Table I (peptide identification scores and sequence coverage are given in supplemental Table S1). The shotgun data have been deposited to the ProteomeXchange Consortium (proteomecentral.proteomexchange.org) via the PRIDE partner repository (27Vizcaíno J.A. Côté R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Pérez-Riverol Y. Reisinger F. Ríos D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1596) Google Scholar) with the data set identifier PXD000879 and DOI 10.6019/PXD000879.Table 1Shotgun identifications of 9 mouse skin biopsies using a Ge-LC-MS/MS strategy (25 bands). Biopsies were collected 5 days (D+5) or 7 days (D+7) after the inoculation of Borrelia burgdorferi ss 297. Each sample was analyzed once and the identifications were performed according to two search engines (Mascot and OMSSA) We developed two LC-SRM methods, M1 and M2. The first method (M1) monitored 33 peptides corresponding to nine Borrelia proteins. The second method (M2) was focused on four Borrelia proteins only but was extended for OspC variants and flagellin (76 monitored peptides). For M1, 33 proteotypic peptides were selected for nine targeted proteins (OspC, flagellin, DbpA, GAPDH, RpoN, BB0081, BBP42, HSP 90, and sensory transduction histidine kinase), and isotopically labeled equivalent peptides were purchased (32 crude PEPotecs peptides and one high purity AQUA peptide (GPNLTEISK, OspC). For M2, 76 proteotypic peptides were selected for only four (OspC, flagellin, DbpA, and GAPDH) among the nine initially targeted proteins in M1, and isotopically labeled equivalent peptides were purchased (74 crude PEPotecs peptides and two high purity AQUA peptides (GPNLTEISK, OspC; ANLGAFQNR, flagellin). For the lower limits of quantification and detection determination (LLOQ and LLOD, respectively), a dilution series of the peptides was realized by spiking crude peptides at different dilution values (1:10,000 to 1:10) and the high purity peptides at different concentrations (500 amol/μl to 50 fmol/μl) in a blank mouse skin matrix and injected in triplicate on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific). The area under curve of all transitions for each peptide were summed and drawn versus the peptide concentration. According to the LLOQ of each peptide for both methods, we separated all the crude peptides into several groups of different dilutions to prepare a concentration-balanced mixture of heavy labeled peptides. Four picomoles of the GPNLTEISK (OspC) and 4 pmol of the ANLGAFQNR (flagellin) AQUA peptides were added to this mixture with a final concentration of 25 fmol/μl. For both methods, nano-LC-MS/MS analysis of the mixture of isotopically labeled peptides afforded a representative MS/MS spectrum for each peptide. Four transitions corresponding to the most abundant y monocharged ions with an m/z value above the doubly charged precursor m/z and four most abundant transitions (if different) were selected from the fragmentation spectrum. Four to eight transitions were monitored for both endogenous and heavy labeled peptides. Thus, a total of 314 transitions corresponding to 66 precursors and nine proteins were measured in M1. A total of 758 transitions corresponding to 152 precursors and four proteins were measured in M2. For the SRM analyses, 1 μl of a mixture of heavy labeled peptides was added to a volume of sample solution containing 2 μg on average (50 μg of protein/25 bands gel) and 6.6 μg of peptides for the gel-based and the gel-free strategies, respectively. All separations were carried out on an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific). For each analysis, the sample was loaded into a trapping column (ZORBAX SB MicroBore Guard (5 μm, 1.0 × 17 mm), Agilent) at 50 μl/min with aqueous solution containing 0.1% (v/v) HCO2H and 2% ACN. After 3 min of trapping, the column was put on line with an Acclaim PepMap RSLC column (15 cm × 300 μm inner diameter, C18, 3 μm, 100 Å; Thermo Fisher Scientific). Peptide elution was performed at 5 μl/min by applying a mixture of solvents A/B. Solvent A was water with 2% ACN and 0.1% (v/v) HCO2H, and solvent B was ACN with 2% water and 0.1% (v/v) HCO2H. Separations were performed by applying three gradients either (i) a linear gradient from 8 to 30% solvent B over 25 min followed by a washing step (3 min at 80% solvent B) and an equilibration step (7 min at 8% solvent B) for the gel bands of both mouse and human skin biopsies H1, H2, H3, H4 and for the liquid digested mouse skin samples; (ii) a linear gradient from 2% to 35% solvent B over 90 min followed by a washing step (5 min at 80% solvent B) and an equilibration step (10 min at 2% solvent B) for the liquid digested human skin sample H1; or (iii) a linear gradient from 5 to 30% solvent B over 57 min followed by a washing step (1 min at 80% solvent B) and an equilibration step (20 min at 5% solvent B) for the gel bands of human skin samples H5 and H6. SRM analyses were performed using the TSQ Vantage triple quadrupole mass spectrometer. The isolation width for both Q1 and Q3 was set to 0.7 m/z unit. The collision gas pressure in Q2 was set at 1.5 millitorr argon. For M1, the collision energy was calculated using the optimized formula CE = 0.03 × m/z + 2.905 for doubly charged precursor ions provided by the supplier. For M2, the collision energy was experimentally optimized by testing nine values centered on the calculated value from the previous formula. Both time-scheduled SRM methods targeted the pairs of isotopically labeled peptides/endogenous peptides in ±2.5-min (except ±1.5-min for human skin sample H4) retention time windows by monitoring a minimum of three transitions for each peptide within a cycle time of 3 s (the target peptides and transitions are given in supplemental Table S2). Mass data collected during LC-SRM were processed with the Skyline open source software package 2.0.9 (28MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. 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• W2099950291 created "2016-06-24" @default.
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• W2099950291 date "2015-05-01" @default.
• W2099950291 modified "2023-10-13" @default.
• W2099950291 title "Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*" @default.
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