Protein Extraction, Enrichment and MALDI MS and MS/MS Analysis from Bitter Orange Leaves (Citrus aurantium)

Abstract

Citrus aurantium is a widespread tree in the Mediterranean area, and it is mainly used as rootstock for other citrus. In the present study, a vacuum infiltration centrifugation procedure, followed by solid phase extraction matrix-assisted laser desorption ionization tandem mass spectrometry (SPE MALDI MS/MS) analysis, was adopted to isolate proteins from leaves. The results of mass spectrometry (MS) profiling, combined with the top-down proteomics approach, allowed the identification of 78 proteins. The bioinformatic databases TargetP, SignalP, ChloroP, WallProtDB, and mGOASVM-Loc were used to predict the subcellular localization of the identified proteins. Among 78 identified proteins, 20 were targeted as secretory pathway proteins and 36 were predicted to be in cellular compartments including cytoplasm, nucleus, and cell membrane. The largest subcellular fraction was the secretory pathway, accounting for 25% of total proteins. Gene Ontology (GO) of Citrus sinensis was used to simplify the functional annotation of the proteins that were identified in the leaves. The Kyoto Encyclopedia of Genes and Genomes (KEGG) showed the enrichment of metabolic pathways including glutathione metabolism and biosynthesis of secondary metabolites, suggesting that the response to a range of environmental factors is the key processes in citrus leaves. Finally, the Lipase GDSL domain-containing protein GDSL esterase/lipase, which is involved in plant development and defense response, was for the first time identified and characterized in Citrus aurantium.

Keywords: Citrus aurantium; GDSLs; MALDI MS/MS; SPE enrichments; vacuum infiltration centrifugation.

Figure 1

Schematic overview of the workflow.

Figure 2

Linear matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) of the chromatographic fractions 39 and 47.

Figure 3

PANTHER functional classification viewed in pie chart. (A) Protein Class; (B) Biological Process.

Figure 4

The protein sequences of citrus-specific genes were functionally annotated with metabolic information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using KEGG Orthology And Links Annotation (BlastKOALA) program.

Figure 5

Alignment of the six identified GDSL variants with the sequence of A. Thaliana (GDL79_ARATH). MS/MS validated sequences are colored, while the yellow highlight the GDSL motif and the active sites (red amino acids).

Figure 6

Sequence of GDSL from citrus by alignment of MS/MS validated peptides, using as reference sequence A0A067EBP6_CITSI (blue string). In bold is reported the validated sequences, in red is indicated the punctual modifications and in yellow is highlighted the catalytic triad, while the gray highlights the region with the major microheterogeneity of the protein.