Prevalidation of potential protein biomarkers in toxicology using iTRAQ™ reagent technology

Glückmann, Matthias; Fella, Kerstin; Waidelich, Dietmar; Merkel, Dietrich; Kruft, Volker; Kramer, Peter-Jürgen; Walter, Yvonne; Hellmann, Jürgen; Karas, Michael; Kröger, Michaela

doi:10.1002/pmic.200600836

Cited by 41 publications

(23 citation statements)

References 36 publications

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“…Each pooled sample contained 40 µg protein. Protein Reduction, Alkylation and iTRAQ labeling were performed using an iTRAQ Reagent 4 Plex Kit according to standard procedures (Gluckmann et al, 2007). Pooled samples were labeled as described below: iTRAQ isobaric reagents 114, normal colon mucosa of mice in the vehicle control group; 115, colon mucosa of mice in the DSS control group; 116, colon mucosa of mice in the DSS and SNE-treated group; 117, colon mucosa of mice in the DSS and PE-treated group.…”

Section: Lc-ms/ms Analysismentioning

confidence: 99%

Preventive Effects of Spirogyra neglecta and a Polysaccharide Extract against Dextran Sodium Sulfate Induced Colitis in Mice

Taya

Kakehashi²,

Wongpoomchai³

et al. 2016

Asian Pacific Journal of Cancer Prevention

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Ulcerative colitis (UC) results from colonic epithelial barrier defects and impaired mucosal immune responses. In this study, we aimed to investigate the modifying effects of a Spirogyra neglecta extract (SNE), a polysaccharide extract (PE) and a chloroform fraction (CF) on dextran sodium sulfate (DSS)-induced colitis in mice and to determine the mechanisms. To induce colitis, ICR mice received 3% DSS in their drinking water for 7 days. Seven days preceding the DSS treatment, oral administration of SNE, PE and CF at doses of 50, 25 and 0.25 mg/kg body weight (low dose), 200, 100 and 1 mg/kg body weight (high dose) and vehicle was started and continued for 14 days. Histologic findings showed that DSS-induced damage of colonic epithelial structure and inflammation was attenuated in mice pre-treated with SNE, PE and CF. Furthermore, SNE and PE significantly protected colonic epithelial cells from DSS-induced cell cycle arrest, while SNE, PE and CF significantly diminished apoptosis. Proteome analysis demonstrated that SNE and PE might ameliorate DSS-induced colitis by inducing antioxidant enzymes, restoring impaired mitochondria function, and regulating inflammatory cytokines, proliferation and apoptosis. These results suggest that SNE and PE could prevent DSS-induced colitis in ICR mice by protection against and/or aiding recovery from damage to the colonic epithelium, reducing ROS and maintaining normal mitochondrial function and apoptosis.

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Section: Lc-ms/ms Analysismentioning

confidence: 99%

Preventive Effects of Spirogyra neglecta and a Polysaccharide Extract against Dextran Sodium Sulfate Induced Colitis in Mice

Taya

Kakehashi²,

Wongpoomchai³

et al. 2016

Asian Pacific Journal of Cancer Prevention

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“…For optimization studies, four aliquots (72 g protein content each) of an MCF-7 (EMEM/insulin culture) protein extract digest solution, spiked with standards after digestion but before SPEC-PTC18 clean-up (see above), were labeled with iTRAQ reagents 114, 115, 116, and 117, and mixed in a ratio of 0.2:1:1:5. Alternatively, different amounts (4,20,20, and 100 g) of the same cell extract were labeled with iTRAQ reagents and mixed in a ratio of 1:1:1:1, to generate the same final protein ratios of 0.2:1:1:5. In addition, three aliquots (5, 25, and 127 g) of a standard protein mix digest solution (0.5 M) were labeled with iTRAQ reagents 114, 116, and 117, and combined 1:1:1 to generate protein ratios of 0.2:1:5.…”

Section: Itraq Labelingmentioning

confidence: 99%

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Armenta

Hoeschele

Lazar

2009

J. Am. Soc. Mass Spectrom.

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An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17␤-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of Ͻ4%. The reproducibility of protein identifications was ϳ60%-67% between duplicate, and ϳ50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P Ͻ 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ϳ2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ϳ16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up-or down-regulated. R ecently, two-dimensional liquid chromatography (2DLC) with tandem MS detection has emerged as an attractive technology for quantitative proteomic profiling of complex cellular extracts [1][2][3][4]. Stable isotope labeling and label-free quantitation strategies have been explored [4 -13]. Isotope labeling approaches rely on the covalent attachment of stable isotope tags to specific amino acid residues of proteins or peptides during metabolic, enzymatic, or chemical processes. Label-free quantitation methods rely on measuring peak areas, intensities, or spectral counts, and benefit from not having to chemically alter the sample. Throughput, however, is lower, and quantitation errors are higher, as the samples are processed independently. Among chemical labeling techniques, isotope tags for relative and absolute quantitation (iTRAQ) has received much attention [14 -30]. In this approach, peptides are labeled with isobaric tags at the N-terminus and the lysine side chains. MS/MS fragmentation produces signature ions that can be used to obtain quantitative information. Perhaps the most attractive advantage of this approach is that it can be used for the simultaneous quantification (relative or absolute) of up to four/eight different samples. Simplicity, of course, is an added benefit.A...

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“…43 Liver fibrosis and cirrhosis are commonly studied using rat models [8] . Among the 44 liver fibrosis animal models, which are induced by bile duct ligation (BDL) [9] , ethanol 45 [10] , carbon tetrachloride (CCl4) [11] or thioacetamide (TAA) [12] , the intraperitoneally 46 TAA-injected rat model is widely used to induce liver fibrosis and cirrhosis and 47 consistently produces liver fibrosis and cirrhosis in rats with a histopathology that is more 48 similar to that of human liver fibrosis and cirrhosis [13][14][15] . In 1948, TAA was first reported 49 as a hepatotoxic agent [16] .…”

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confidence: 99%

“…http://dx.doi.org/10.1101/125120 doi: bioRxiv preprint first posted online Apr. 6, 2017; hepatitis [43] and is one of the potential hepatocarcinogenic biomarkers [44] . patients, which suggests that DJ-1 maybe a candidate prognostic biomarker of HCC [45] .…”

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confidence: 99%

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

Zhang

Yuan

et al. 2017

Preprint

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Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis, which results from the deposition of extracellular matrix (ECM) components, is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide (TAA) liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS). A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins in the TAA 1-week, 3-week, 6-week and 8-week groups. A total of 40 urinary proteins were statistically significant (fold change >2 and p<0.05), 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase (ALT) and aspartate transaminase (AST) changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Masson’s staining.

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Prevalidation of potential protein biomarkers in toxicology using iTRAQ™ reagent technology

Cited by 41 publications

References 36 publications

Preventive Effects of Spirogyra neglecta and a Polysaccharide Extract against Dextran Sodium Sulfate Induced Colitis in Mice

Preventive Effects of Spirogyra neglecta and a Polysaccharide Extract against Dextran Sodium Sulfate Induced Colitis in Mice

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

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