Ab 5800 maldi tof tof mass spectrometry

The main protein spots on the 2-DE gel of 6M roots were manually excised from 2-DE gels and subjected to perform an in-gel digestion with the modified bovine trypsin (Cat. 11418025001, Roche, Basel, Switzerland) as described [64 (link)]. After digestion, mass spectra of trypsin-digested peptides map fingerprinting were obtained on an AB 5800 MALDI-TOF/TOF mass spectrometry (AB SCIEX, Foster City, MA, USA) with a laser wavelength of 349 nm as described [2 (link)]. Ten main spectra were selected for MS/MS analysis and searched for a self-constructed database derived from the original TKS genome. The nucleotide and protein sequences were deposited in the Genome Warehouse (GWH; http://bigd.big.ac.cn/gwh/) under the accession number PRJCA000437 (https://academic.oup.com/nsr), which includes 46,731 gene sequences [16 (link)]. Protein matches were considered as a positive identification with a Mascot score higher than 75 and at least two matched peptides, meanwhile individual ion score > 31 (p < 0.05). Detailed information for the identified proteins is provided as the supplementary data (Table S1 and Figure S2).

After the target protein spots were cut out, the in-gel digestion was performed as described [8 (link)]. After enzymatic digestion, these peptides were collected and identified by the AB 5800 MALDI-TOF/TOF mass spectrometry (MS) instrument (AB SCIEX, Foster City, CA, USA). The ProteinPilot Software (Version 4.5) and a Mascot Algorithm (version 2.3) were used to search against the Hevea genome scaffolds (BioProject ID: PRJNA80191, www.ncbi.nlm.nih.gov/nuccore/448814761) and the draft genome (GenBank: AJJZ01000000) with 46,718 sequences and 17,435,757 residues [63 (link)]. The parameters were set as: precursor tolerance 300 ppm, 0.3 Da tolerance as MS/MS fragment, trypsin as the enzyme, carbamidomethylation as the fixed modification (C) and oxidation (M) as the variable modification. Detailed information on all identified protein spot mass searches can be found in Supplemental Figure S1 and Table S1.