Computational protocol: Identification of T. gondii Myosin Light Chain-1 as a Direct Target of TachypleginA-2, a Small-Molecule Inhibitor of Parasite Motility and Invasion

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Protocol publication

[…] All mass spectrometry preparations were performed in the VGN Proteomics Facility at the University of Vermont. Dimethyl labelling was adapted from protocols described previously , . Briefly, lyophilized tryptic peptides were dissolved in 50 µL of 1 M HEPES-NaOH, pH 7.5, and incubated at 25°C for 10 min with 4 µL each of freshly made 4% (v/v) d0-formaldehyde in H2O and 600 mM sodium cyanoborohydride (NaCNBH3) in 1 M NaOH, or 4% (v/v) d2-formaldehyde in H2O and 600 mM sodium cyanoborodeuteride (NaCNBD3) in 1 M NaOH for “light” and “heavy” labelling, respectively. Samples were incubated for an additional 10 min with a second round of labelling reagents, followed by quenching of the reaction with 50 µL of 10% (v/v) trifluoroacetic acid and incubation at 25°C for 1 h. The light and heavy labelled samples were mixed and desalted using PepClean C18 spin columns (Thermo Fisher Scientific, Rockford, IL). Spin columns were prewashed twice with 200 µL Buffer B (99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid) and equilibrated with two washes of 200 µL 0.1% (v/v) formic acid. The sample was passed over the column twice to ensure maximal binding to resin, eluted with two rounds of 30 µL Buffer B and dried in a SpeedVac (Thermo Fisher Scientific, San Jose, CA) at 25°C for 2 h.Mass measurements were made in an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) with a liquid chromatography interface set up as previously described . Precursor scans (360–1600 m/z) were conducted in the Orbitrap at 30,000 resolution followed by ten data-dependent MS/MS scans in the LTQ linear ion trap on the most abundant ions identified in the precursor scan. Dynamic exclusion was enabled with a repeat count of three for a duration of 30 s. The lock mass feature for internal calibration was enabled and set to calibrate on the mass of a polydimethylcyclosiloxane ion ([(Si(CH3)2O)5 + H+]+, m/z  =  371.10120) , .MS/MS spectra were manually examined for the presence of characteristic “marker” b- and y-ions (b5-ion m/z = 564.2, b7-ion m/z = 692.3; y4-ion m/z = 687.3, y6-ion m/z = 903.4) calculated using the MS-Product utility program of ProteinProspector v. 5.10.11 (http://prospector.ucsf.edu/prospector/mshome.htm; accessed 2013 Dec 10), for the initial identification of the tachypleginA-2-induced, modified V46-R59 peptide. To collect targeted, high mass accuracy MS/MS spectra, the fragmentation method described above was revised to perform the MS/MS scans in the Orbitrap instead of the LTQ linear ion trap, with a normalized collision energy of 35, activation Q of 0.25, activation time of 30 s and an isolation m/z width of 1.2. One MS/MS scan targeted the m/z = 849.35115 peak corresponding to that of the modified peptide for fragmentation. MS3 analysis was performed with the precursor scan in the Orbitrap followed by MS/MS and MS3 scans in the LTQ linear ion trap, with the MS3 scan targeting the m/z = 1006.40950 peak corresponding to the y7-ion.SEQUEST analysis of tandem mass spectra was conducted using the TgMLC1 amino acid sequence, requiring no enzyme specificity, allowing a 20 ppm window around the precursor mass and allowing differential mass additions of 15.99491 on methionines for oxidation, 79.96633 on serines, threonines and tyrosines for phosphorylation, and the following on cysteines: 0 for a free sulfhydryl, 57.02146 for carbamidomethylation, 71.03711 for acrylamide adduction and 225.11859 for compound-induced modification. For the dimethyl-labelled datasets, a static increase of 28.0313 was set on N-termini and lysines, in addition to a dynamic modification of 6.03766 on N-termini and lysines for heavy labelled peptides. […]

Pipeline specifications

Software tools Protein Prospector, Comet
Application MS-based untargeted proteomics
Organisms Toxoplasma gondii, Homo sapiens
Diseases Leukemia, T-Cell