Scout-MRM: multiplexed targeted mass spectrometry-based assay without retention time scheduling exemplified by Dickeya dadantii proteomic analysis during ...
Supporting Information
Scout-MRM: multiplexed targeted mass spectrometry-based assay without retention time scheduling exemplified by Dickeya dadantii proteomic analysis during plant infection Blandine Rougemont1, Sébastien Bontemps Gallo2,5, Sophie Ayciriex1, Romain Carrière1, Hubert Hondermarck3, Jean Marie Lacroix2, J.C. Yves Le Blanc4, and Jérôme Lemoine1,* 1
Université de Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut des Sciences Analytiques, CNRS UMR 5280, 5 rue de la Doua, F-69100 Villeurbanne, France. 2
Université de Lille, CNRS, UMR 8576 Unité de Glycobiologie Structurale et Fonctionnelle, F 59000 Lille, France. 3
School of Biomedical Sciences & Pharmacy and Hunter Medical Research Institute, University of Newcastle, Callaghan, NSW 2308, Australia. 4
Sciex, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada.
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Present address: Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA * To whom correspondance should be addressed Description of Supplementary Information Materials and Methods: pages S2-S3 Figure S1. Illustration of Scout-MRM independence from retention time shift: page S4 Figure S2. Venn diagram illustrating the number and percentage of peptide recovered after Scout-MRM method transfer: page S5 Figure S3. Proposed two–injection workflow to help for optimal peptide recovery when transferring or implementing Scout-MRM on a column with different C18 stationary phase: page S6 Table S1 (Excel file). Sequences of the 19 scout peptides sorted by elution time. Table S2 (Excel file). List of identified Dickeya dadantii proteins in the discovery experiment and Skyline library. Table S3 (Excel file). Complete Scout-MRM method used to analyze the Dickeya dadantii proteome samples extracted at 0h (liquid culture) and 24, 48 and 72 post-infection of chicory leaves. Table S4 (Excel file). KEEG (Kyoto Encyclopedia of Genes and Genomes) classification and quantification of Dickeya dadantii proteins at 24, 48 and 72h post-infection of chicory leaves.
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Materials and Methods Bacterial growth and infection Dickeya dadantii EC3937 wild-type strain was grown overnight in 100 mL of lysogeny broth (LB) medium at 28°C until stationary growth phase. Before infection, 50 mL of bacterial cell culture was recovered by centrifugation (4,000g for 30 min) and bacterial pellet was lyophilized (data point of the time course: 0h). For infection, 1 mL of cell culture was recovered by centrifugation and suspended in physiological water to 109 bacteria/mL. After wounding of chicory (Cichorium intybus) leaves, the plants were inoculated with 107 bacteria and incubated in a dew chamber at 28°C. After 24h, 48h and 72h, whole soft rots were collected from either 20, 12 or 8 chicory leaves for normalizing the bacteria number, suspended in 40mL of physiological saline solution and vortexed for 1 min. Bacteria were filtered to discard plant residue - the filtrate was centrifuged (4,000g for 30 min) and the bacterial pellet was lyophilized. Lyophilized bacteria were resuspended in 15 mL of lysis buffer containing 6M guanidine in Tris-HCl buffer and disrupted by sonication for 30 min. The protein concentration of the extracts was determined in triplicate by Bradford assay (Rothi®Quant, Carl Roth). Sample preparation for proteomic measurements 500 µL of bacterial cell lysate (about 500 µg proteins) were submitted to protein digestion procedure. Proteins were reduced with 16.5 mM dithiothreitol for 40 min at 60°C and alkylated with 45 mM iodoacetamide for 40 min at room temperature, in the dark. Samples were diluted by 4 mL of 50 mM NH4HCO3 to decrease guanidine concentration (< 1mM) before adding porcine trypsin (Sigma) at a final enzyme/substrate ratio of 1/25 (w/w). Trypsin digestion was conducted at 37°C overnight and was stopped by acidification with formic acid. The peptide mixtures were sonicated and centrifuged 15 min at 15,000 g before being loaded onto an OASIS® HLB 3 cc (60 mg) cartridges (Waters) that was desalted according to the manufacturer instructions and eluted with 100 % methanol supplemented with 0.5 % formic acid. 100 µL of 10 % glycerol in methanol were added to prevent peptide adsorption during evaporation under nitrogen flux at 40°C. Peptides were re-suspended in 190 µL of water/methanol (90:10, v/v) containing 0.5 % formic acid. Before LC-MS/MS analysis, samples were supplemented by 30 µL of a stock solution (water/methanol (90:10, v/v) containing 0.5 % formic acid) containing the scout peptides, each at the concentration of 500 ng/mL. Shotgun approach using DDA acquisition mode. Peptide samples were subjected to liquid chromatography using an HPLC surveyor MS pump system equipped with two columns in series XB-C18 Aeris™ Peptide (3.6 µm, 150 mm x 2.1 mm, Phenomenex). Peptide elution was performed by applying a mixture of solvent A and B. Solvent A was HPLC grade water with 0.1 % (v/v) formic acid, and solvent B was HPLC grade acetonitrile with 0.1 % (v/v) formic acid. After a washing step (5 min at 5 % solvent B), separation was performed using a gradient of 5 % to 25 % acetonitrile in 0.1 % formic acid over 115 min (120 min total method length) at a flow rate of 300 µL/min. 15 µL of each sample was injected in duplicates. The HPLC system was directly coupled with a QExactive mass spectrometer. For ionization, 4,000 V voltage and 500°C capillary temperature were used. The DDA method consisted of a full MS scan (35k resolution, 3e6 automatic gain control (AGC) target, 250ms injection time, 400 to 1250 m/z) followed by up to 10 data-dependent MS/MS acquisitions on the top 10 most intense precursor ions (17.5k resolution, 1e6 AGC target, 150ms injection time, 2 m/z isolation window, 28% normalized energy collision, centroid mode).
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DDA data processing and analysis Raw DDA data files were converted to mzXML and searched using Protein Prospector (version 5.17.0) against the Dickeya dadantii 3937 Uniprot database (4,500 entries non reviewed, UniprotKB, 2016.5.30) allowing for variable oxidation of methionine, Nterminal acetylation and pyroglutamate. Carbamidomethylation of cysteines was set as a fixed modification. Trypsin digestion allowing no missed cleavages was selected. Precursor mass tolerance was set to 20 ppm, and fragment ion tolerance to 25 ppm. Identified peptides with an FDR precursor ion. The exported transitions lists were monitored in the sample used in the DDA experiments. Only the 2 high-responding peptides detected per protein were then added to Dickeya dadantii library.
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Figure S1. Screenshot of the “Build Acquisition Method” of provisional Scout-MRM patch software page derived from Advanced Scheduled MRM software. As depicted, no retention time is specified for the targeted Scout and endogenous peptides. Primary and secondary Scout transitions are labeled with 1 and 2 while the transitions of endogenous peptides are only labeled with 4.
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Figure S2. Illustration of Scout-MRM independence from retention time shifts. The digested proteome of D dadantii extracted 72h post-infection was analyzed using two different Scout-MRM methods in order to mimic, for instance, a difference of void volume at the LC device level. Upper chromatogram was obtained with the following gradient: a washing step of 5 min at 95% of solvent A (HPLC grade H2O with 0.1 % (v/v) formic acid) and 5% of solvent B (HPLC grade CH3CN with 0.1 % (v/v) formic acid), followed by a separation performed using a 5% to 25% gradient of solvent B over 115 min (120 min total method length) at a flow rate of 300 µL/min. The same gradient was used for the lower chromatogram, except that there was a 10 min washing step. The inserts show reconstructed ion chromatograms for identical transitions of groups detected in both gradients. Despite different retention times, all targeted peptides were detected.
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Figure S3. Dynamic range of detected peptides at 0, 24, 48 and 72h plant infection.
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Figure S4. Venn diagram illustrating the number and percentage of peptide recovered after Scout-MRM method transfer. The digested proteome of D dadantii extracted after 24h post-infection was diluted twice and distributed in two vials for subsequent ScoutMRM analysis on two distinct LC-MS/MS systems (two Agilent 1290 chromatographs coupled to either a 6500 or a 5500 hybrid triple quadrupole/linear quadrupole ion trap mass spectrometer), while keeping the same C18 reverse-phase column. Without any adjustment, a peptide recovery of 94% between the two LC-MS/MS systems was calculated after data integration using Skyline. Only 6% of all peptides (i.e. 31 peptides out of 519) were detected specifically with the most sensitive instrument (6500 Qtrap).
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Figure S5. Proposed two–injection workflow to help for optimal peptide recovery when transferring or implementing Scout-MRM. Some peptides exhibit selectivity difference (i.e. retention time shift) following the change of C18 reverse phase chemistry or when moving to a chromatographic device with significantly different void volume/flow ratio (i.e. conventional to nanoflow systems for instance). We thus designed a simple workflow aimed to facilitate assay transfer and implementation between laboratories that do not use similar chromatographic formats. The first step, the initial Scout-MRM (1) is carried out in order to pinpoint the peptides not affected by selectivity issue, i.e. those remaining in their initially affiliated transition group. In a second run (2), only the peptides not previously detected are monitored after their positioning both in upstream and downstream transition groups. The final method (3) is then ultimately built by merging the couples of transition groups (i.e. bracketed by the same scout peptides) with all successfully detected peptides.
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Table S1 (Excel file): Sequences of the 19 scout peptides sorted by elution time. For each scout peptide, the table reports the sequence, the name of the corresponding protein and its Uniprot accession number (www.uniprot.org). The m/z values of “precursor ion” and “product ion” for each scout transition signal are indicated in column E and F, respectively. The last column provides the number of transitions triggered by each scout peptides to track D.dadantii peptides. Table S2 (Excel file): List of identified Dickeya dadantii proteins in the discovery experiment and Skyline library. Worksheets 1 and 2 provide the peptide sequences and names of proteins identified using Protein Prospector (see Methods section “DDA data processing and analysis”) for 0 and 48h proteome extracts, respectively. The worksheet 3 corresponds to the final Skyline library after selection of peptides and fragment ions (see Methods section “Creation of Scout-MRM method: selection of targeted proteins, peptides and transitions”). Table S3 (Excel file): Complete Scout-MRM method used to analyze the Dickeya dadantii proteome samples extracted at 0h (liquid culture) and 24, 48 and 72 post-infection of chicory leaves. The table contains the final Scout-MRM method used to monitor the 782 peptides in the digested proteome extracts (the 0h reference culture and the 24, 48 and 72h post-infection chicory leaves). Since the actual Scout-MRM software patch is a modified version of the “advanced scheduled MRM” software available with Analyst 1.6.2®, “Time”, “Window” and “Dwell weight” columns have to be filled by 0, 1 and 1, respectively, to run a Scout-MRM experiment. Then, for each of the 19 scout peptides, two transitions were monitored, named type 1 (1) and type 2 (2) in the column “Ion Type”. The most intense transition, named “type 1”, corresponds to the triggering-scout signal, which is associated to a threshold value defined in the “Threshold” column. All the transitions monitoring D.dadantii peptides are named type 4 (4). Finally, all type 4 transitions are affiliated to a scout group, named by the first three letters of the scout amino acid sequence. The table contains also all other parameters required during any MRM experiment building, such as the m/z values of the precursor, the product ions, and optimized voltages: *DP: Declustering Potential, **EP: Entrance Potential, ***CE: Collision Energy, ****CXP: Cell eXit Potential. Table S4 (Excel file): KEEG (Kyoto Encyclopedia of Genes and Genomes) classification and quantification of Dickeya dadantii proteins at 24, 48 and 72h post-infection of chicory leaves. The table contains the quantification results of D.dadantii proteins measured by ScoutMRM, and are related to Figure 3b. After peptide validation (see Methods section “ScoutMRM data analysis”), the proteins identified in the 0h (liquid culture), 24, 48, and 72h postinfection proteome extracts (375 proteins) were classified by biological function according to KEGG classification 1,2. Fold changes in protein relative quantities are expressed relative to 0h (liquid culture). The selected proteins used to build the heatmap (Figure 3b) are highlighted in grey. References: 1. Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. & Tanabe, M. Nucleic Acids Res. 44, D457–62 (2016). 2. Kanehisa, M. & Goto, S. Nucleic Acids Res. 28, 27–30 (2000).
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