Analysis of dynamic protein carbonylation in rice embryo during germination through AP‐SWATH

Abstract

Seed germination is an important aspect of the plant life cycle, during which, reactive oxygen species (ROS) accumulate. The accumulation of ROS results in an increase in protein oxidation of which carbonylation is the most canonical one. However, there is insufficient information concerning protein oxidation, especially carbonylation and its contribution to seed germination. In this study, biotin hydrazide labeled chromatography combined with sequential window acquisition of all theoretical fragment ion spectra (SWATH) method was used to analyze the dynamic pattern of protein carbonylation in rice embryos during germination. A total of 1872 unique proteins were quantified, among which 288 carbonylated peptides corresponding to 144 proteins were determined based on the filtering through mass shifts of modified amino acids. In addition, 66 carbonylated proteins were further analyzed based on their carbonylation intensity in four stages of germination. These identified carbonylated proteins were mainly involved in maintaining the levels of ROS, abscisic acid and seed reserves. Remarkably, a peroxiredoxin was found with 23 unique carbonylated peptides, and the expression of which was consistent with its increased activity. This study describes the dynamic pattern of carbonylated proteins during seed germination, and may help to further understand the biochemical mechanisms on this process.

Mitochondrial Proteome Studies in Seeds during Germination

“…While the general identification of mitochondrial proteins in seeds was performed using both gel-based (2D-PAGE) and MS-based proteomic approaches (LC-MS/MS, iTRAQ, SRM) (Table S1), the identification of PTMs (such as carbonylation and phosphorylation) of mitochondrial proteins in dry and in germinating seeds has been dominated by in-depth MS-based studies [40,41,42,43] (Table S2). Zhang et al [40] utilized biotin hydrazide labeled chromatography, which allows enrichment of carbonylated proteins, combined with the sequential window acquisition of all theoretical fragment ion spectra (SWATH) method to analyze the protein carbonylation pattern in rice embryos isolated from different stages of seed germination (Table S2).…”

“…Zhang et al [40] utilized biotin hydrazide labeled chromatography, which allows enrichment of carbonylated proteins, combined with the sequential window acquisition of all theoretical fragment ion spectra (SWATH) method to analyze the protein carbonylation pattern in rice embryos isolated from different stages of seed germination (Table S2). To identify phosphorylated proteins in germinating seeds (rice and maize), different phosphopeptide enrichment methods, such as strong-cation exchange (SCX) or polymer-based metal ion affinity capture (PolyMAC) were implemented, followed by analyses using either shotgun approach and LC-MS/MS, or nano-liquid chromatography coupled with tandem mass spectrometry (nano-LC-MS) [41,42,43] (Table S2).…”

“…Here, we present a list of carbonylated proteins identified so far in Arabidopsis and rice embryo mitochondria with the 2,4-dinitrophenylhydrazine (DNPH) immunoassay [61] and biotin hydrazide labeling-affinity chromatography combined with the SWATH quantitative method [40] (Table S2). The latter approach, applied for the first time in plants, provided more comprehensive analysis on the dynamics of protein carbonylation during seed germination and let to the identification of more and new carbonylated proteins than previous 2-DE based studies [60,63].…”

“…Despite the antioxidant functions of MSD and Prx [70,71] and the protective role of chaperones, which probably act as a shield protecting other proteins against oxidative damage [72], it seems that these proteins are relatively susceptible to carbonylation and under prolonged oxidative stress they could become dysfunctional [69]. Among the detected carbonylated proteins, the TCA cycle and OXPHOS enzymes, such as aconitate hydratase 2 (ACO2), isocitrate dehydrogenase, succinate dehydrogenase, as well as the beta subunit of ATP synthase were found in Arabidopsis and rice seeds [40,61]. These proteins might be rapidly inactivated during oxidative stress mainly because of their location in proximity of the ROS-generating sites [73].…”

Proteomics (SWATH-MS) informed by transcriptomics approach of tropical herbPersicaria minorleaves upon methyl jasmonate elicitation

“…SWATH-MS analysis is capable of profiling any given proteome quantitatively with better coverage than a traditional shotgun proteomics (which mostly relies on data-dependent acquisition) (Gillet et al, 2012). Several studies using this approach have successfully profiled proteomes from human (Bourassa et al, 2015; Chang et al, 2015), animals (Li et al, 2016; Perez-Patiño et al, 2016), microbes (Loke et al, 2016b) and plants (Zhang et al, 2016; Zhu et al, 2016). SWATH-MS proteomics is also known to be a far more sensitive, reproducible and accurate than the Western blotting alone (Gillet et al, 2012; Picotti & Aebersold, 2012).…”