Proteomic Identification of Cellular Protease Substrates Using Isobaric Tags for Relative and Absolute Quantification (iTRAQ)

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. Methyl jasmonate (MeJA), a plant hormone, mediates diverse developmental process and defense responses which induce a variety of metabolites. In plants, little is known about autophagy-mediated responses against MeJA. In this study, we used high-throughput comparative proteomics to identify proteins of latex in the laticifers. The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS proteomics were performed, and 298 proteins among MeJA treated groups and the control group of Euphorbia kansui were identified. It is interesting to note that 29 significant differentially expressed proteins were identified and their associations with autophagy and ROS pathway were verified for several selected proteins as follows: α-L-fucosidase, β-galactosidase, cysteine proteinase, and Cu/Zn superoxide dismutase. Quantitative real-time PCR analysis of the selected genes confirmed the fact that MeJA might enhance the expression of some genes related to autophagy. The western blotting and immunofluorescence results of ATG8 and ATG18a which are two important proteins for the formation of autophagosomes also demonstrated that MeJA could promote autophagy at the protein level. Using the electron microscope, we observed an increase in autophagosomes after MeJA treatment. These results indicated that MeJA might promote autophagy in E. kansui laticifers; and it was speculated that MeJA mediated autophagy through two possible ways: the increase of ROS induces ATG8 accumulation and then aotophagosome formation, and MeJA promotes ATG18 accumulation and then autophagosome formation. Taken together, our results provide several novel insights for understanding the mechanism between autophagy and MeJA treatment. However, the specific mechanism remains to be further studied in the future.

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Comparative Proteomic Analysis of Dipsacus asperoides Roots from Different Habitats in China

Molecules ◽

10.3390/molecules25163605 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3605

Author(s):

Haijun Jin ◽

Hua Yu ◽

Haixia Wang ◽

Jia Zhang

Keyword(s):

Proteomic Analysis ◽

Absolute Quantification ◽

Pcr Analysis ◽

Allene Oxide ◽

Allene Oxide Cyclase ◽

Protein Levels ◽

Depth Analysis ◽

Differential Proteins ◽

Isobaric Tags

Dipsacus asperoides is a kind of Chinese herbal medicine with beneficial health properties. To date, the quality of D. asperoides from different habitats has shown significant differences. However, the molecular differences in D. asperoides from different habitats are still unknown. The aim of this study was to investigate the differences in protein levels of D. asperoides from different habitats. Isobaric tags for relative and absolute quantification (iTRAQ) and 2DLC/MS/MS were used to detect statistically significant changes in D. asperoides from different habitats. Through proteomic analysis, a total of 2149 proteins were identified, of which 42 important differentially expressed proteins were screened. Through in-depth analysis of differential proteins, the protein metabolism energy and carbohydrate metabolism of D. asperoides from Hubei Province were strong, but their antioxidant capacity was weak. We found that three proteins, UTP-glucose-1-phosphate uridylyltransferase, allene oxide cyclase, and isopentyl diphosphate isomerase 2, may be the key proteins involved in dipsacus saponin VI synthesis. Eight proteins were found in D. asperoides in response to environmental stress from different habitats. Quantitative real-time PCR analysis confirmed the accuracy and authenticity of the proteomic analysis. The results of this study may provide the basic information for exploring the cause of differences in secondary metabolites in different habitats of D. asperoides and the protein mechanism governing differences in quality.

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Quantitative proteomic analysis of G-protein signalling inStagonospora nodorumusing isobaric tags for relative and absolute quantification

PROTEOMICS ◽

10.1002/pmic.200900474 ◽

2010 ◽

Vol 10 (1) ◽

pp. 38-47 ◽

Cited By ~ 20

Author(s):

Tammy Casey ◽

Peter S. Solomon ◽

Scott Bringans ◽

Kar-Chun Tan ◽

Richard P. Oliver ◽

...

Keyword(s):

G Protein ◽

Proteomic Analysis ◽

Absolute Quantification ◽

Quantitative Proteomic Analysis ◽

G Protein Signalling ◽

Isobaric Tags

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Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis

Disease Markers ◽

10.1155/2015/196589 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Bhavna Daswani ◽

Manoj Kumar Gupta ◽

Shubhangi Gavali ◽

Meena Desai ◽

Gajanan J. Sathe ◽

...

Keyword(s):

Absolute Quantification ◽

Osteoclast Formation ◽

Mineral Density ◽

Monocyte Migration ◽

Receptor Activator ◽

Lower Chamber ◽

Isobaric Tags ◽

First Time ◽

Upper Chamber ◽

Increased Susceptibility

Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n=80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P<0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P<0.0001) and this was additive in combination with RANKL (receptor activator ofNFkBligand) placed in the lower chamber (P=0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

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iTRAQ-Based Comparative Proteomics Analysis of Urolithiasis Rats Induced by Ethylene Glycol

BioMed Research International ◽

10.1155/2020/6137947 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Yanan Cao ◽

Bin Duan ◽

Xiaowei Gao ◽

E. Wang ◽

Zhitao Dong

Keyword(s):

Ethylene Glycol ◽

Kidney Stones ◽

Enrichment Analysis ◽

Absolute Quantification ◽

Rat Models ◽

Protein Protein Interaction ◽

Parallel Reaction Monitoring ◽

High Prevalence ◽

Isobaric Tags ◽

And Control

Nephrolithiasis is a frequent chronic urological condition with a high prevalence and recurrence rate. Proteomics studies on urolithiasis rat models are highly important in characterizing the pathophysiology of kidney stones and identifying potential approaches for preventing and treating kidney stones. The isobaric tags for relative and absolute quantification (iTRAQ) were performed to identify differentially expressed proteins (DEPs) in the kidney between urolithiasis rats and control rats. The results showed that 127 DEPs (85 upregulated and 42 downregulated) were identified in urolithiasis and control rats. The functions of DEPs were predicted by Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein–protein interaction (PPI) network analysis. The expression of four upregulated proteins (Tagln, Akr1c9, Spp1, and Fbn1) and four downregulated proteins (Hbb, Epb42, Hmgcs2, and Ca1) were validated by parallel reaction monitoring (PRM). Proteomics studies of ethylene glycol-induced urolithiasis rat models using iTRAQ and PRM helped to elucidate the molecular mechanism governing nephrolithiasis and to identify candidate proteins for the treatment of kidney stones.

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Proteomic Analysis of Beef Tenderloin and Flank Assessed Using an Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)

Animals ◽

10.3390/ani10010150 ◽

2020 ◽

Vol 10 (1) ◽

pp. 150

Author(s):

Zhaomin Lei ◽

Jianping Wu ◽

Deyin Zhang ◽

Ting Liu ◽

Shengguo Zhao ◽

...

Keyword(s):

Proteomic Analysis ◽

Expression Patterns ◽

Tca Cycle ◽

Absolute Quantification ◽

Absolute Quantitation ◽

Oxidation Reduction ◽

The Tca Cycle ◽

Isobaric Tag ◽

Isobaric Tags

Herein, we performed a proteomic analysis of tenderloin and flank steaks from Simmental cattle using the isobaric tags for a relative and absolute quantification (iTRAQ) approach. We identified 17 amino acids in both steaks, and Gly, Cys, Ile, Lys, and Pro differed most in abundance between the steak types (p < 0.05). A comparison of the expression patterns in steaks revealed 128 differentially expressed proteins (DEPs), of which 44 were up-regulated and 84 were down-regulated. Furthermore, 27 DEPs (p < 0.05) were subjected to gene ontology (GO) analysis, and many were found to be related to oxidation-reduction, metabolism, hydrogen ion transmembrane transport, transport, the tricarboxylic acid (TCA) cycle, mitochondrial electron transport, and the conversion of nicotinamide adenine dinucleotide (NADH) to ubiquinone. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also implicated these DEPs in various signalling pathways, including oxidative phosphorylation, cardiac muscle contraction, the TCA cycle, biosynthesis, and the metabolism. These findings provide a new insight into key proteins involved in the determination of amino acid composition in beef.

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GZFLW Induces Apoptosis of Ectopic Endometrial Stromal Cells via Promoting VPS53 Protein Stability

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/1293630 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10

Author(s):

Ziyu Zhang ◽

Faying Liu ◽

YunYun Xu ◽

Huang Huang ◽

Yang Zou ◽

...

Keyword(s):

Molecular Mechanism ◽

Stromal Cells ◽

In Vitro Study ◽

Absolute Quantification ◽

Endometrial Stromal Cells ◽

Gynecological Diseases ◽

Isobaric Tags ◽

Differential Expression Protein ◽

Vacuolar Protein

Endometriosis is still a major problem in obstetrics and gynecology. While GZFLW (Gui Zhi Fu Ling Wan) has been originally used for treating gynecological diseases, however, the molecular mechanism that GZFLW acts on endometriosis is not clear. To investigate the molecular mechanism that GZFLW plays role on endometriosis, iTRAQ (isobaric tags for relative and absolute quantification) proteomics and human endometrial stromal cells (Y14) obtained from a patient with endometriosis were used in in vitro study. Our results demonstrated that GZFLW decreased Y14 cells proliferation while increased cells apoptosis. The differential expression protein VPS53 (Vacuolar protein sorting 53 homolog) was predicted by iTRAQ coupled LC-MS/MS and further identified by western blot. Besides, GZFLW induced VPS53 protein level by promoting its stabilization. Our findings highlight a novel role for VPS53 in gynecology and provide a potent therapeutic strategy against endometriosis.

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