Pepseq

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 ].

Separation and sequencing of tryptic peptides with liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using a Q-Tof Premier (Jasco International) coupled with nanoACQUITY UPLC® (Waters). Peptide fragments were applied onto a nanoACQUITY BEH C18 100 µm × 100 mm column, and eluted at a flow rate of 0.4 µl/min for 30 min using a 3–40% linear gradient of solvent B of 0.1% HCOOH in CH₃CN and 60–97% linear gradient of solvent A of 0.1% HCOOH in water. Analysis was performed using a positive ion mode at 3 kV capillary voltage. The mass range was set from 350 to 1700 m/z, and the MS/MS spectra were acquired for the peaks with at least 15 counts. The spectra were processed using ProteinLynx v4.1 software (Waters) and MASCOT (www.matrixscience.com) database searches of the NCBInr database for all Viridiplantae sequences. Peptides that could not be identified in the database were identified as partial sequences by de novo sequences using PepSeq (Waters). In this case, homologous proteins were predicted using BPASTP (DDBJ, http://www.ddbj.nig.ac.jp/welcome-j.html). Proteins were identified by the assignment of at least three peptide fragments, but proteins of less than 21 kDa were assigned using at least two peptide fragments. Proteins were predicted with homology of more than 70% using at least three peptides.