Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes

Abstract

Proteomic analysis of upland cotton was performed to profile the global detectable proteomes of ovules and fibers using two-dimensional electrophoresis (2DE). A total of 1,203 independent protein spots were collected from representative 2DE gels, which were digested with trypsin and identified by matrix-assisted laser desorption and ionization-time-offlight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The mass spectrometry or tandem mass spectrometry (MS or MS/MS) data were then searched against a local database constructed from Gossypium hirsutum genome sequences, resulting in successful identification of 975 protein spots (411 for ovules and 564 for fibers). Functional annotation analysis of the 975 identified proteins revealed that ovule-specific proteins were mainly enriched in functions related to fatty acid elongation, sulfur amino acid metabolism and post-replication repair, while fiber-specific proteins were enriched in functions related to root hair elongation, galactose metabolism and D-xylose metabolic processes. Further annotation analysis of the most abundant protein spots showed that 28.96% of the total proteins in the ovule were mainly located in the Golgi apparatus, endoplasmic reticulum, mitochondrion and ribosome, whereas in fibers, 27.02% of the total proteins were located in the cytoskeleton, nuclear envelope and cell wall. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of the ovule-specific protein spots P61, P93 and P198 and fiber-specific protein spots 230, 477 and 511 were performed to validate the proteomics data. Protein-protein interaction network analyses revealed very different network cluster patterns between ovules and fibers. This work provides the largest protein identification dataset of 2DE-detectable proteins in cotton ovules and fibers and indicates potentially important roles of tissue-specific proteins, thus providing insights into the cotton ovule and fiber proteomes on a global scale. cotton, 2DE, MALDI-TOF/TOF, GO enrichment, protein-protein interaction networks Citation:Jin, X., Wang, L., He, L., Feng, W., and Wang, X. (2016). Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes. Sci China Life Sci 59, 154 -163.

Genome-Wide Identification and Expression Analysis of the Ascorbate Oxidase Gene Family in Gossypium hirsutum Reveals the Critical Role of GhAO1A in Delaying Dark-Induced Leaf Senescence

“…Total RNA was extracted from different tissues (fibers at different development stages, roots, stems, leaves, petals, and anthers) using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized from 1 µg of total RNA using the TIANScript RT Kit (Tiangen, Beijing, China), and then used as a template for qRT-PCR analysis using gene-specific primers (Supplementary Table S4), with the Gossypium hirsutum ubiquitin 7 (GhUBQ7) gene as the internal control [7,67].…”

Two-Dimensional Gel Electrophoresis-Based Proteomic Analysis Reveals N-terminal Truncation of the Hsc70 Protein in Cotton Fibers In Vivo

“…In recent years, gel-free high-throughput MS technologies have been widely used instead of 2-DE gels, benefitting from their high sensitivity and high throughput for identifying proteins78910111213. However, 2-DE gel technology can still provide advantages that are not available for gel-free approaches, such as visualization maps of protein profiles, information about the MW and pI of individual protein spots and, most importantly, reliable evidence for existing protein isoforms14. This work describes the application of 2-DE gel technology to investigate the post-translational regulation of functional proteins.…”

“…Gel-free high-throughput mass spectrometry (MS) approaches have been applied in recent years to identify proteins on a larger scale with higher sensitivity and reveal new aspects of the protein-level regulatory mechanism of cotton fiber development910111213. However, 2-DE technology is irreplaceable because it yields visualization maps of protein profiles, which provide information on the abundance of proteins and reliable evidence for existing protein isoforms14.…”

Comparative Proteomic Investigation of Plasma Reveals Novel Potential Biomarker Groups for Acute Aortic Dissection

“…Mass spectra-(MS-) based technologies have been greatly developed and are the most widely used proteomic approaches for their high throughput and sensitivity [19][20][21]. However, classic two-dimensional gel electrophoresis-based proteomic technologies are still irreplaceable since they provide a visualization map of the proteome and the truncation or modification information of protein isoforms [22][23][24]. Isobaric tags for relative and absolute quantitation (iTRAQ) technology is a high-efficiency nongel quantitative proteomic assay particularly suitable to identify biomarkers in low-abundance proteins [25][26][27][28].…”