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Dimethyl labeling-based quantitative PTM proteomic analysis of drought-regulated phosphoproteomes in soybean drought-tolerant cultivar

ABSTRACT: The isotopic dimethyl labeling-based quantitative post-translational modification proteomics was applied to study the phosphoproteomic changes associated with the drought responses of two contrasting soybean cultivars. A total of 9,457 non-redundant, unambiguous, label-independent and repeatable phosphopeptides were subsequently identified from six experimental replicates, corresponding to 9,234 of deduced unique phosphoproteins. These soybean proteins contain a total of 20,880 phosphosites, 84.9% of which were found to be novel phosphosites of the soybean phosphoproteome. The overly post-translationally modified proteins is 2.05% of the phosphoproteins identified. Most of these extensively phosphorylated proteins are photoreceptors, mRNA-, histone- and phospholipid-binding as well as serine/threonine/tyrosine protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of the phosphosite of phosphoprotein follows the exponential decay law, Y=4.13e^(-0.098X)-0.04. From 218 significantly regulated unique phosphopeptide groups, 188 significantly regulated phosphoproteins were found, and they are enriched in biological functions of water transport and deprivation, methionine metabolic process, photosynthesis/light reaction, and response to cadmium ion, osmotic stress and ABA under drought treatment. A total of 15 and 20 drought-tolerant cultivar significantly regulated phosphoproteins are transcription factors and protein kinases/phosphatases, respectively. More than 50% of these phosphoproteins are considered to be novel regulatory components, presumably mediating the development of the soybean drought tolerance under water deprivation process. The association of five randomly selected phosphoproteins with the drought response was corroborated with the qRT-PCR method.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Glycine Max

TISSUE(S): Plant Cell

SUBMITTER: Shuaijian DAI

LAB HEAD: Ning, Li

PROVIDER: PXD022806 | Pride | 2023-05-10

REPOSITORIES: Pride

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Project description:Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10 % additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

Dataset Information

Dimethyl labeling-based quantitative PTM proteomic analysis of drought-regulated phosphoproteomes in soybean drought-tolerant cultivar

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