Dynamic N-glycoproteome analysis of maize seedling leaves during de-etiolation using Concanavalin A lectin affinity chromatography and a nano-LC–MS/MS-based iTRAQ approach

Abstract

The identification of N -glycosylated proteins with information about changes in the level of N -glycosylation during de-etiolation provides a database that will aid further research on plant N -glycosylation and de-etiolation. N-glycosylation is one of the most prominent and abundant protein post-translational modifications in all eukaryotes and in plants it plays important roles in development, stress tolerance and immune responses. Because light-induced de-etiolation is one of the most dramatic developmental processes known in plants, seedlings undergoing de-etiolation are an excellent model for investigating dynamic proteomic profiles. Here, we present a comprehensive, quantitative N-glycoproteomic profile of maize seedlings undergoing 12 h of de-etiolation obtained using Concanavalin A (Con A) lectin affinity chromatography enrichment coupled with a nano-LC-MS/MS-based iTRAQ approach. In total, 1084 unique N-glycopeptides carrying 909 N-glycosylation sites and corresponding to 609 proteins were identified and quantified, including 186 N-glycosylation sites from 162 proteins that were significantly regulated over the course of the 12 h de-etiolation period. Based on hierarchical clustering analysis, the significantly regulated N-glycopeptides were divided into seven clusters that showed different N-glycosylation patterns during de-etiolation. We found no obvious difference in the enriched MapMan bincode categories for each cluster, and these clustered significantly regulated N-glycoproteins (SRNPs) are enriched in miscellaneous, protein, cell wall and signaling, indicating that although the N-glycosylation regulation patterns of these SRNPs might differ, they are involved in similar biological processes. Overall, this study represents the first large-scale quantitative N-glycoproteome of the model C4 plant, maize, which is one of the most important cereal and biofuel crops. Our results greatly expand the maize N-glycoproteomic database and also shed light on the potential roles of N-glycosylation modification during the greening of maize leaves.

Large-scale Proteomic and Phosphoproteomic Analysis of Maize Seedling Leaves During De-etiolation

“…To perform a deep analysis of the maize proteome and phosphoproteome during de-etiolation, the first leaves of etiolated 7-day-old maize seedlings (B73 inbred) were illuminated with white light and harvested at 0 h, 1 h, 6 h and 12 h for mass spectrometry analysis ( Figure 1 ). We chose these samples for analysis because in our previous studies [25, 31, 32] we found that the first leaves of 7-day-old etiolated maize seedlings showed no significant changes in growth compared with light-grown seedlings when subjected to illumination for 12 hours, and few proteins involved in development and growth were found to be differently expressed when comparing the proteomes of etiolated and normal leaves. Moreover, the etiolated leaves turned green after 12 hours of illumination, which indicated that processes related to greening in leaves were completed.…”

“…The maize inbred line B73 was used in this study. The seedlings were planted and samples were collected as described previously [32]. Under the same conditions, two biological replicates were performed and the first seedling leaves from each replicate were rapidly sampled.…”

“…Total proteins were extracted from maize seedling leaves using a 10% (w:v) trichloroacetic acid (TCA)/acetone solution as described previously [32]. The protein concentration of each sample was determined using the 2-D Quant kit (GE Healthcare).…”

“…Protein extraction of two sets of maize samples (0 h, 1 h, 6 h, and 12 h, ∼5 mg each) and trypsin digestion were performed as previously described [32]. According to the manufacturer’s instructions, samples were labeled with iTRAQ 4plex reagent (ABSciex, MA, US) and then combined.…”

“…A Dionex RSLC system interfaced with QExactive HF (ThermoFisher, San Jose, CA) was used to carried out HPLC-MS/MS primarily. Due to instrument availability, 2D-HPLC-MS/MS and phosphor-proteomic samples were analyzed using a Dionex RSLC system interfaced with a Velos LTQ Orbitrap ETD (ThermoFisher, San Jose, CA) as described previously [32]. To achieve the mass spectrometry data, a data-dependent acquisition procedure with a cyclic series of full scans was used and acquired in the Orbitrap with a resolution of 120,000 (QExactive HF) or 60,000 (VELSO LTQ Orbitrap ETD).…”