2D Mini-Gel Proteome Profiling and MS Identification

A preparative 2D-mini gel (7 cm IPG strip, GE Healthcare, Chicago, IL, USA) stained Coomassie (Brilliant Blue G, Sigma Aldrich, Darmstadt, Germany), containing an equal amount of each non-labeled individual sample mixed with 10 µg of IS labeled sample, in a total of 75 µg of protein, was performed for further spot cut-off for mass spectrometry (MS) protein identification. The introduction of some labeled sample into preparative gel facilitates gel match with analytical gels for spot location and picking for MS analysis. The protein spot of interest was cut off from the gel and trypsin digested, as we previously described [16 (link),17 (link)]. Tryptic peptides, suspended in 50% (v/v) ACN and 0.1% (v/v) trifluoroacetic acid (TFA, for HPLC, ≥99.0%, Sigma Aldrich, Darmstadt, Germany) and submitted to sonication in an ultrasonic bath during 15 min, were directly applied on a 100-well matrix-assisted laser desorption/ionization (MALDI) plate with 5 mg/mL α-cyano-4-nydroxycinnamic acid (α-CHCA, 1:1) prepared in 0.1% TFA/60% ACN (Merck, Darmstadt, Germany) (v/v) and allowed to co-crystallize at RT. Peptides were analysed on an Applied Biosystems SCIEX 4800 Plus MALDI Proteomics Analyser with time-of-flight/time-of-flight (TOF/TOF) ion optics exactly as described [18 (link)]. The identified proteins are shown in Table S2 (Supplementary Materials).