Maldi tof tof mass spectrometer

Mass spectrometry (MS) was performed at the Yonsei Proteome Research Center (YPRC, Korea). Peptides obtained after trypsin digestion from cell lysate were analyzed with a MALDI-TOF/TOF mass spectrometer (4800 ABSciex, USA). For sample preparation, 70% ACN was allowed to flow through the porous tip to remove impurities attached to the resin. Next, a solution of 2% formaldehyde was passed through the column to create acidic conditions. Following this pretreatment, the samples to be analyzed were passed through the column. Subsequently, the wash buffer (2% FA) was used to remove impurities such as salt and chemicals. Finally, the peptides attached to the resin were elute.

Aliquots of muscle homogenate (25 μg per sample) were solubilized in Laemmli sample buffer (BioRad Laboratories, Inc., CA, USA), denatured by boiling at 100°C for 5 min and separated by SDS-PAGE. Molecular weight standards (Precision Plus Protein All Blue Standards, BioRad) were also run on each gel to identify the proteins molecular weights. One-dimensional gels were stained by Coomassie Brilliant Blue R-250 (BioRad), and the stained gel images were semiquantitatively analyzed using the Image Studio Lite 3.1.4 software for Macintosh (LI-COR Biosciences, NE, USA).
Bands of interest were manually excised and sent for identification by peptide mass fingerprint to the Inbiotec S.L. (León, Spain) proteomics laboratory, where the samples were processed and analyzed with a 4800 Proteomics Analyzer matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer (ABSciex, MA, USA) according to the previously described methods of Oliván et al [25 ]. A database search on Mascot Generic Files combining MS and MS/MS spectra was performed using Mascot v 2.2 from Matrix Science through the Global Protein Server v 3.6 (ABSciex). When the Mascot score was greater than 85 points, the identified protein was considered a valid candidate.

Differentially expressed protein spots identified in 2D-DIGE were excised automatically through Ettan spot picker (GE Healthcare, USA) from the preparative gel and subjected to in-gel digestion of the proteins as reported earlier (32 (link)). Briefly, the gel pieces were reduced with DTT and alkylated with IAA and subjected to enzymatic digestion using trypsin (100 ng) by keeping at overnight incubation at 37°C. The tryptic peptides from in-gel digestion were extracted and spotted on a MALDI plate with CHCA matrix (10 mg/ml). Protein identification was performed using a 4,800 MALDI-TOF/TOF mass spectrometer (AB Sciex, USA) linked to a 4,000 series explorer software (v.3.5.3) equipped with Nd: YAG 355 nm laser and a repetition rate of 200. The mass range of 800 to 4,000 Da was used, and the mass spectra were acquired in reflector mode with 20 kV and 18 kV as acceleration and extraction voltages, respectively. MS/MS spectra were acquired for the 12 most abundant precursor ions, with a total accumulation of 2,500 laser shots and collision energy of 2 kV. The MASCOT version 2.1 (http://www.matrixscience.com) was used for the data analysis by keeping the taxonomy as Homo sapiens, database as SwissProt, enzyme as trypsin, oxidation of methionine as variable and carbamidomethylation of cysteine as a fixed modification. The MS mass tolerance was set as 75 ppm and MS/MS as 0.4 Da.