Mtp anchorchip 400 384 target plate

The band corresponding to the enzyme identified as ACPase II was excised from polyacrylamide gels and digested with trypsin Gold-Mass V582A (Promega), according to Schevchenko et al [39 (link)]. The resulting peptides of each spot were submitted to mass spectrometric analyses using an UltraFlexIII MALDI-TOF/TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight), controlled with Flex Control 3.0 software (Bruker Daltonik). The sample was mixed with α-cyano-4hydroxycinnamic acid matrix solution (3:1, v/v) directly applied onto an MTP AnchorChip 400/384 target plate (Bruker Daltonik) and dried at room temperature. Peptides presenting mono isotopic masses were obtained in reflector mode with external calibration using the Protein Calibration Standard (Bruker Daltonik). Peptide MS/MS spectra were obtained by means of LIFT fragmentation. The software Flex Analysis 3.0 (Bruker Daltonik) and PepSeq (Waters) were used for mass spectrometric data analysis. Peptide primary structures were inferred by means of manual interpretation of fragmentation. The obtained sequences were then searched against the NCBI_nr protein database (www.ncbi.nlm.nih.gov) using the algorithm Blastp. The identified protein sequences were analyzed using the Peptide Mass tool from the ProtParam Server (www.expasy.org) in order to predict theoretical molecular weight [40 ].

15 × 10⁶ CD4+ T cells from healthy controls were treated with 10% HC/SF serum and incubated for 18 h. Total protein extraction was conducted using RIPA buffer with a 1× protease inhibitor cocktail. 40 µg of protein extract was separated on a 10% acrylamide gel at 100 V for 90 min. Samples were digested with 0.02 μg/μL trypsin and ammonium bicarbonate for 24 h at 37 °C. The peptides were then desalted and concentrated by ZipTipC18 technology (Millipore, Billerica, MA, USA) with double-distilled water containing 80% acetonitrile and 0.1% trifluoroacetic acid. The eluate was spread onto a matrix-assisted laser desorption/ionization (MALDI) target plate (MTP AnchorChip 400/384 target plate, Bruker Daltonic, Bremen, Germany) with α-cyano-4-hydroxycinnamic acid used as the matrix. Subsequently a MALDI–time of flight (TOF)/TOF MS (Ultraflex III; Bruker Daltonic) MS was performed.
A database search (Swiss-Prot) using the Mascot 2.2 search engine (Matrix Science Inc., Boston, MA, USA) and Bruker Bio-Tool 3.2 software (Bruker Daltonics, Bremen, Germany) was performed with the calibrated and annotated spectra to calculate the peptide mass signal for each entry into the sequence database, to compare the experimental MALDI-MS and MALDI-MS/MS dataset, and to assign a statistical weight to each individual peptide match using empirically determined factors. Adapted/rewritten from [31 (link)].