Identification of flagellin peptides in N. benthamiana apoplastic fluid
Purified flagellin from Pseudomonas Pta6605 WT was supplemented with N. benthamiana apoplastic fluids from plants transiently expressing only p19 or p19 and bGAL1 (a galactosidase). By performing subsequent LC/MSMS we wanted to identify and quantify the levels of flagellin peptides released by endogenous peptidases in the AF.
Sample Processing Protocol
Sample generation for MS. After incubation the samples were supplemented with 4 Vol MS-grade Acetone, incubated on ice for 1h and then centrifuged at 18000 ×g for 15 min. 4/5th of the supernatants were then transferred to fresh Eppendorf tubes and the acetone evaporated by vacuum centrifugation. The dried peptide sample was then taken up in 0.1% formic acid and immediately analysed by LC/MS. LC-MS/MS. Experiments were performed on an Orbitrap Elite instrument (Thermo, Michalski et al., 2012) that was coupled to an EASY-nLC 1000 liquid chromatography (LC) system (Thermo). The LC was operated in the one-column mode. The analytical column was a fused silica capillary (75 µm × 35 cm) with an integrated PicoFrit emitter (New Objective) packed in-house with Reprosil-Pur 120 C18-AQ 1.9 µm resin (Dr. Maisch). The analytical column was encased by a column oven (Sonation) and attached to a nanospray flex ion source (Thermo). The column oven temperature was adjusted to 45 °C during data acquisition. The LC was equipped with two mobile phases: solvent A (0.1% formic acid, FA, in water) and solvent B (0.1% FA in acetonitrile, ACN). All solvents were of UHPLC (ultra high performance liquid chromatography) grade (Sigma). Peptides were directly loaded onto the analytical column with a maximum flow rate that would not exceed the set pressure limit of 980 bar (usually around 0.5 – 0.8 µL/min). Peptides were subsequently separated on the analytical column by running a 140 min gradient of solvent A and solvent B (start with 7% B; gradient 7% to 35% B for 120 min; gradient 35% to 80% B for 10 min and 80% B for 10 min) at a flow rate of 300 nl/min. The mass spectrometer was operated using Xcalibur software (version 2.2 SP1.48). The mass spectrometer was set in the positive ion mode. Precursor ion scanning was performed in the Orbitrap analyzer (FTMS; Fourier Transform Mass Spectrometry) in the scan range of m/z 350-1800 and at a resolution of 60000 with the internal lock mass option turned on (lock mass was 445.120025 m/z, polysiloxane, Olsen et al., 2005). Product ion spectra were recorded in a data dependent fashion in the ion trap (ITMS; Ion Trap Mass Spectrometry) in a variable scan range and at a rapid scan rate. The ionization potential (spray voltage) was set to 1.8 kV. Peptides were analyzed using a repeating cycle consisting of a full precursor ion scan (3.0 × 106 ions or 50 ms) followed by 15 product ion scans (1.0 × 104 ions or 50 ms) where peptides are isolated based on their intensity in the full survey scan (threshold of 500 counts) for tandem mass spectrum (MS2) generation that permits peptide sequencing and identification. CID (collision-induced dissociation) collision energy was set to 35% for the generation of MS2 spectra. During MS2 data acquisition dynamic ion exclusion was set to 120 seconds with a maximum list of excluded ions consisting of 500 members and a repeat count of one. Ion injection time prediction, preview mode for the FTMS, monoisotopic precursor selection and charge state screening were enabled. Only charge states >1 were selected for fragmentation.
Data Processing Protocol
RAW spectra were submitted to an Andromeda (Cox et al., 2011) search in MaxQuant (version 188.8.131.52) using the default settings (Cox & Mann, 2008). Label-free quantification and match-between-runs was activated (Cox et al., 2004). MS/MS spectra data were searched against the p19_vector_proteins.fasta (2 entries) and ACE_0383_SOI_v02.fasta (containing the sequence of flagellin and bGAL1). All searches included also a contaminants database (as implemented in MaxQuant, 267 sequences). The contaminants database contains known MS contaminants and was included to estimate the level of contamination. Andromeda searches allowed oxidation of methionine residues (16 Da) and acetylation of protein N-terminus (42 Da) as dynamic modification. Enzyme specificity was set to “unspecific”. The instrument type in Andromeda searches was set to Orbitrap and the precursor mass tolerance was set to ±20 ppm (first search) and ±4.5 ppm (main search). The MS/MS match tolerance was set to ±0.5 Da. The peptide spectrum match FDR and the protein FDR were set to 0.01 (based on target-decoy approach). Minimum peptide length was 8 amino acids and the maximum length 25. For protein quantification unique and razor peptides were allowed. Modified peptides were allowed for quantification. The minimum score for modified peptides was 40.
Buscaill P, Chandrasekar B, Sanguankiattichai N, Kourelis J, Kaschani F, Thomas EL, Morimoto K, Kaiser M, Preston GM, Ichinose Y, van der Hoorn RAL. Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides. Science. 2019 364(6436) PubMed: 30975858