4800 plus maldi tof tof analyser

Total proteins from the roots and leaves were extracted with acetone–trichloracetic acid (Černý et al., 2011 (link); Baldrianová et al., 2015 (link)); leaf extracts were subsequently depleted of the majority of the Rubisco protein using the IgY/Rubisco Spin Column. Proteins were resolved by 2-DE (Černý et al., 2011 (link), 2014 ), and the spot pattern was analysed using Decodon Delta 2D software (http://www.decodon.com). Responses to stimuli (HS/modulated CK pool) of proteins corresponding to detected spots were deemed significant if there was an absolute stimulated/mock spot volume ratio ≥1.35, with t-test P value <0.05, and similar profiles in two biological replicates (three technical replicates per sample). Selected protein spots were digested with trypsin and analysed using a 4800 Plus MALDI TOF/TOF analyser (AB Sciex; Nd:YAG laser 355nm), and the resulting spectra were searched by local Mascot v. 2.1 (Matrix Science) against the TAIR10 Arabidopsis database (for details see Černý et al., 2013 (link); Baldrianová et al., 2015 (link)).

MALDI-TOF (matrix-assisted laser desorption/ionisation-time of flight) MS was carried out on all glycopeptides used in this study. HPLC peak fractions were diluted 1:1 with 3,4-diaminobenzophenone dissolved at 10 mg/mL in 75% (v/v) acetonitrile or with 2,5-dihydroxybenzoic acid dissolved at 20 mg/mL in 70% (v/v) methanol. The glycopeptide/matrix mix (1 µL) was then spotted on a 384-well target plate (AB Sciex) and left to dry at room temperature overnight. The following day samples were analysed in positive or negative-ion mode using a 4800 Plus MALDI–TOF/TOF Analyser (AB Sciex) with 4000 Series Explorer (Applied Biosystems). Tandem MS of glycopeptides was carried out using collision-induced dissociation fragmentation. Data were extracted with the Data Explorer software (Applied Biosystems), and semi-manual data analysis was carried out with the open-source software Skyline (MacCoss Lab). For final graphic editing, CorelDraw software was used.