Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars

Abstract

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e−0.098X − 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINβ1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

Editing of rice (Oryza sativa L.) OsMKK3 gene using CRISPR/Cas9 decreases grain length by modulating the expression of photosystem components

“…DEPs were submitted to the STRING database (version 11.5) (http://string.embl.de) to perform protein interaction analysis with a moderate confidence (0.4) and construct a putative protein‐protein interaction network. The network was then exported as a tsv file and visualized using Cytoscape (Version 3.9.1) to construct volvox graphic [57]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository [58, 59] with the dataset identified PXD038056.…”

Exhaustive Cross-Linking Search with Protein Feedback

“…Last, we arbitrarily picked five high confident spectra from Beveridge et al 36 and put these five spectra together with other 6,636 irrelevant cross-linking spectra from soybean data set. 44 We used ECL-PF to identify them with the setting in Supplemental Table S8. ECL-PF successfully reported these five spectra.…”

Exhaustive Cross-linking Search with Protein Feedback

“…We observe from different datasets that, among the valid CSMs, the number of proteins corresponding to the identified peptides is very limited because not every protein can interact with others, nor does every protein have multiple available link sites inside itself. For example, in the Arabidopsis thaliana dataset [9], on average, one protein can have 6.02 different peptide sequences. But, on average, one protein can only have 1.39 unique peptide sequences.…”

“…To optimize the top p choice and proportion restriction, we use different real datasets [9–12] to run ECL-PF with different combinations of p and proportion. During the optimization process, we assume the higher the number of CSMs, the better the parameters.…”