Mascot1

Proteins were isolated and analyzed by slight modification of a previously described procedure (Brzychczy-Włoch et al., 2013c (link)). Briefly, bacteria were cultured on brain heart infusion broth (Biocorp) at 37°C for 24 h. After centrifugation, they were suspended directly in the buffer for electrophoresis according to Heilmann et al. (1996) (link). This made it possible to isolate moonlighting proteins and preserve their native structure. Protein concentration was analyzed using the Lowry’s method (Lowry et al., 1951 (link)). The immunoreactive proteins were identified by immunoblotting within a complex protein mixture that has been fractionated using discontinuous gradient polyacrylamide gel (5, 7.5, 10, and 12.5%). Individual reactive spots were cut out from gels and submitted to tryptic digestion, then analyzed by mass spectrometry (spectrometer LC-MS/MS Orbitrap, Thermo). Proteins were identified by Mascot¹ (Matrix Science, London, United Kingdom) and statistical methods.

Images (300 dpi, 16-bit grayscale pixel depth) were scanned using UMAX Powerlook 2100XL (UMAX, Taipei, China) and were analyzed using Image Master 2D Platinum Software (Version 5.0, GE Healthcare) as described by Hajduch et al. (2007) (link). Protein spots showing a significant difference in intensity (P < 0.05) of greater than 1.5-fold were selected and manually excised from the gel for protein identification. Mass spectrometry (MS) analysis of selected protein spots was performed by Shanghai Applied Protein Technology Co., Ltd. (Shanghai, China), using the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) method (Hajduch et al., 2007 (link)). The MS/MS data were searched against the ‘plant’ subset of the NCBI non-redundant sequence database using Mascot¹ (Matrix Science).