List of PKL and mzML files generated by LC-MS/MS for identification of amaranth and quinoa seed storage proteins
Description
Amaranth and quinoa seed proteins were extracted by a polarity-based method. Hydrophilic and hydrophobic proteins were analysed by 2-DE, and the representative spots for each species were identified by LC-MS/MS. AH1, hydrophilic amaranth proteins; AH0, hydrophobic amaranth proteins; CH1, hydrophilic quinoa proteins; CH0, hydrophobic quinoa proteins.
Files
Steps to reproduce
Samples were digested using mass spectrometry-grade Trypsin Gold (Promega, Madison, WI, USA) in 5 mM AB. The hydrolysis was carried out overnight at 37 °C, and the resulting peptides were extracted three times by dehydration with 100% acetonitrile (ACN) and rehydrated with 1% formic acid (FA) with centrifugation at 1000 × g for 30 s. A final step of dehydration with 100% ACN was then performed. Finally, peptides were dried using a CentriVap (Labconco), solubilised in 1% FA, and desalted with ZipTip-C18 (Merck Millipore, Darmstadt, Germany). Peptides from 2-DE spots were fractionated by nanoscale liquid chromatography with a nanoACQUITY UPLC System (Waters, Milford, MA, USA) equipped with a Symmetry C18 precolumn (5 μm, 20 mm × 180 μm, Waters) and a BEH130 C18 (1.7 μm, 100 mm × 100 μm, Waters) analytical column. The lock mass compound, [Glu1]-Fibrinopeptide B (Sigma-Aldrich), was delivered by the auxiliary pump of the nanoACQUITY UPLC System at 200 nL/min at a concentration of 100 fmol/mL to the reference sprayer of the Nano-Lock-Spray source of the mass spectrometer. Mass spectrometric analysis was performed using a SYNAPT-HDMS Q-TOF (Waters). The spectrometer was operated in V-mode, and analyses were performed in positive mode ESI. The TOF analyser was externally calibrated with [Glu1]-Fibrinopeptide B at 50-2422 m/z. The data were lock-mass corrected post-acquisition using the doubly protonated monoisotopic ion of [Glu1]-Fibrinopeptide B. The reference sprayer was sampled every 30 s. The RF applied to the quadrupole was adjusted to efficiently transmit ions of 50−2000 m/z. MS and MS/MS spectra were acquired in alternating low-energy and elevated-energy modes of acquisition (MSe), and MS/MS spectrum data sets were used to generate PKL files with the Protein Lynx Global Server v2.4 (Waters). Proteins were then identified using PKL files with the MASCOT search engine v2.5 (Matrix Science). Identifications were performed against the A. hypochondriacus (v1.0 and v2.1) [22,23] and C. quinoa [24] proteome databases, available at https://phytozome-next.jgi.doe.gov/ and https://plants.ensembl.org/index.html. Trypsin specific protease with two missed cleavages allowed was established; carbamidomethyl cysteine (+57.021 Da), methionine oxidation (+15.995 Da), asparagine/glutamine/arginine deamidation (+0.984 Da) and serine/threonine/tyrosine phosphorylation (+79.966 Da) were set as dynamic modifications; mass tolerance for precursor and fragment ions was set to 20 ppm and 0.1 Da, respectively. Identifications were considered valid only for peptide matches with at least two MS/MS spectra matched at a 99% confidence level with identity or extensive homology statistically significant at p < 0.01 and above an identity threshold for Fold Discovery Rate (FDR) ≤ 1%.