Dataset Information

An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics.

ABSTRACT: The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics.

SUBMITTER: Nguyen TV

PROVIDER: S-EPMC8460067 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Rice panicle traits serve as critical indicators of both yield potential and germplasm resource quality. However, traditional manual measurements of these traits, which typically involve threshing, are not only laborious and time-consuming but also prone to introducing measurement errors. This study introduces a high-throughput and nondestructive method, termed extraction of panicle traits (EOPT), along with the software Panicle Analyzer, which is designed to assess unshaped intact rice panicle traits, including the panicle grain number, grain length, grain width, and panicle length. To address the challenge of grain occlusion within an intact panicle, we define a panicle morphology index to quantify the occlusion levels among the rice grains within the panicle. By calibrating the grain number obtained directly from rice panicle images based on the panicle morphology index, we substantially improve the grain number detection accuracy. For measuring grain length and width, the EOPT selects rice grains using an intersection over union threshold of 0.8 and a confidence threshold of 0.7 during the grain detection process. The mean values of these grains were calculated to represent all the panicle grain lengths and widths. In addition, EOPT extracted the main path of the skeleton of the rice panicle using the Astar algorithm to determine panicle lengths. Validation on a dataset of 1,554 panicle images demonstrated the effectiveness of the proposed method, achieving 93.57% accuracy in panicle grain counting with a mean absolute percentage error of 6.62%. High accuracy rates were also recorded for grain length (96.83%) and panicle length (97.13%). Moreover, the utility of EOPT was confirmed across different years and scenes, both indoors and outdoors. A genome-wide association study was conducted, leveraging the phenotypic traits obtained via EOPT and genotypic data. This study identified single-nucleotide polymorphisms associated with grain length, width, number per panicle, and panicle length, further emphasizing the utility and potential of this method in advancing rice breeding.

Project description:Proteins, as executives of genes' instructions, are responsible for cellular phenotypes. Integratingproteomics with gene microarray, we conducted this study to identify potential protein biomarkers of colorectal cancer (CRC). Isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS) was applied to screen and identify differentially expressed proteins between paired CRC and adjacent normal mucosa. Meanwhile, Affymetrix U133plus2.0 microarrays were used to perform gene microarray analysis. Verification experiments included immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay (ELISA) of selected proteins. Overall, 5469 differentially expressed proteins were detected with iTRAQ-MS from 24 matched CRC and adjacent normal tissues. And gene microarray identified 39859 differential genes from 52 patients. Of these, 3083 differential proteins had corresponding differentially expressed genes, with 245 proteins and their genes showed >1.5-fold change in expression level. Gene ontology enrichment analysis revealed that up-regulated proteins were more involved in cell adhesion and motion than down-regulated proteins. In addition, up-regulated proteins were more likely to be located in nucleus and vesicles. Further verification experiments with IHC confirmed differential expression levels of 5 proteins (S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1) between CRC and normal tissues. Besides, western blot showed a stepwise increase of annexin A3 abundance in normal colorectal mucosa, adenoma and CRC tissues. ELISAresults revealed significantly higher serum levels of S100 calcium-binding protein A9 and annexin A3 in CRC patients than healthy controls, validating diagnostic value of these proteins. Cell experiments showed that inhibition of annexin A3 could suppress CRC cell proliferation and aggressiveness. S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1 were probably potential biomarkers of colorectal cancer. Annexin A3 was a potentially valuable therapeutic target of CRC.

Dataset Information

An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets