5800 mass spectrometer

Candidates for protein identification were selected based on fold-change comparisons between control and blast samples and the calculated p-value. Spots of interest were selected using the Ettan Spot Picker (GE Healthcare) and in-gel digested with Trypsin Gold (Promega, Madison, WI, USA). The tryptic peptides were desalted with ZipTip C18 (Millipore, Billerica, MA, USA) and eluted with 5 mg/ml α-cyano-4-hydroxycinnamic acid in 25 mM ammonium bicarbonate, 50% acetonitrile and 0.1% trifluoroacetic acid and spotted onto an AB SCIEX MALDI plate (AB Sciex, Framingham, MA, USA). MALDI-TOF MS and TOF/TOF (tandem MS/MS) were performed using a 5800 mass spectrometer (AB Sciex, Concord, Ontario, Canada). The peptide mass and associated fragmentation data were determined using the GPS Explorer Workstation equipped with a MASCOT search engine (Matrix Science, Boston, MA, USA). Searches were performed without constraining protein molecular weight or isoelectric point, with variable methionine oxidation and cysteine carbamidomethylation, and with one missed cleavage permitted. Candidates with an ion confidence interval (CI%) or protein score CI% > 95 were considered significant.

MALDI‐TOF (MS) and TOF/TOF (tandem MS/MS) were performed on a 5800 mass‐spectrometer (AB Sciex). MALDI‐TOF mass spectra were acquired in reflectron positive ion mode, averaging 2000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 2000 laser shots per fragmentation spectrum on each of the 5–10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions).

Protein spots that were statistically significant (see Section IEF, SDS-PAGE, Image Scan and Data Analysis) with a p ≤ 0.1 and cut off value of 1.5-fold were picked up using the Ettan Spot Picker (GE Healthcare). The picked gel spots were washed a few times, digested with modified porcine trypsin protease (Trypsin Gold, Promega), and then desalted using Zip-tip C18 (Millipore, Billerica, MA, USA) (Robbins et al., 2013 (link); Gupta et al., 2014 (link); Hayashi et al., 2015 (link); Das et al., in press ). Peptides were eluted from the Zip-tip with 0.5 μl of matrix solution (α-cyano-4-hydroxycinnamic acid, 5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid, and 25 mM ammonium bicarbonate) and spotted onto a MALDI plate (Robbins et al., 2013 (link); Hayashi et al., 2015 (link); Das et al., in press ).
MALDI-TOF (MS) and TOF/TOF (tandem MS/MS) were performed on a 5800 mass spectrometer (AB Sciex). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 2000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 2000 laser shots per fragmentation spectrum on each of the 10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions) (Gupta et al., 2014 (link); Hayashi et al., 2015 (link); Das et al., in press ).