Eight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases

Pierce, Andrew; Unwin, Richard D.; Evans, Caroline A.; Griffiths, Sue; Carney, Louise; Zhang, Liqun; Jaworska, Ewa; Lee, Chia-Fang; Blinco, David; Okoniewski, Michał; Miller, Crispin; Bitton, Danny A.; Spooncer, Elaine; Whetton, Anthony D.

doi:10.1074/mcp.m700251-mcp200

Cited by 242 publications

(209 citation statements)

References 35 publications

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“…was determined to be the significant cutoff threshold (p Ͻ 0.05) for the up-regulated proteins, and reciprocally 0.77 was the cutoff threshold for the down-regulated proteins (data are shown in the supplemental data). Similar cutoff thresholds have been used in other iTRAQ studies (21,22).…”

Section: Methodsmentioning

confidence: 99%

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Tan¹,

Tan

Lin

et al. 2008

Molecular & Cellular Proteomics

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Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a shortchain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrateresponsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer. Molecular & Cellular Proteomics 7:1174 -1185, 2008.In developed countries, colorectal cancer is a prevalent disease with high mortality and morbidity rates (1). This disease has emerged as the top malignancy in Singapore. Environmental factors are responsible for about 80% of the cases, whereas genetic predisposition accounts for the minority 20% of cases. Epidemiological evidence suggests that high intake of dietary fiber reduces the incidence and risk of this neoplasm (2, 3). A wealth of studies has shown that butyrate produced from anaerobic fermentation of indigestible carbohydrate is the molecule responsible for the chemopreventive properties of a fiber-rich diet (4 -6).Although butyrate serves as an energy source for normal colonocytes, in vivo and in vitro studies have shown that at physiological concentrations this natural short-chain fatty acid mediates cell maturation with the promotion of growth arrest followed by differentiation and/or apoptosis of cancer cells (7-11). These biological effects are crucial in colorectal cancer therapy as colonic transformation is characterized by multistage alterations of tissue homeostasis resulting in aberrant cell division and/or cell death (12, 13). Butyrate has been purported as a potential anticancer agent. This initiated notable research in identifying proteins that contribute to its biological effects (14, 15). However, most of these investigations focused on one target at any one time and were thus unable to systematica...

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Section: Methodsmentioning

confidence: 99%

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Tan¹,

Tan

Lin

et al. 2008

Molecular & Cellular Proteomics

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“…Maintaining similar physicochemical properties between differentially labeled peptides is of major importance for the peptide pairs of the same amino acid sequence to be detected simultaneously during LC-MS/MS analysis, and for their respective ionic signals to provide reliable measurements of their relative abundance. The reagents allow simultaneous comparison of up to eight different samples with the eight-channel iTRAQ chemistry (Pierce et al, 2008). SILAC labeling to target Arg and Lys residues can be used in three flavors, and a combination of two sets of three-point time courses possessing a common time point allows to obtain fivepoint time courses.…”

Section: The Necessity Of Semi-quantitative Ms Analysesmentioning

confidence: 99%

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

Pflieger

Gonnet

Bentem

et al. 2011

Mass Spectrometry Reviews

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Proteomes are intricate. Typically, thousands of proteins interact through physical association and post‐translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as “systems” rather than collections of individual protein molecules. The abstraction of the interacting proteome to “protein networks” has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome‐wide physical protein–protein‐binding interactions organized into Protein Interaction Networks (PINs), and proteome‐wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome‐wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein–protein interactions, and MS‐based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state‐of‐the‐art MS‐based analytical pipelines for the purpose to characterize proteome‐scale networks. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:268–297, 2011

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“…With dimethylation labeling, iTRAQ and TMT www.intechopen.com labeling, multiplexing can be achieved, which is not possible for 18 O labeling. iTRAQ is capable of simultaneously analyzing eight samples (Pierce et al, 2008), whereas with TMT labeling, six samples can be measured together (Thompson et al, 2003). It should be noted that the use of commercial isobaric iTRAQ or TMT labels can be cost-prohibitive.…”

Section: Chemical and Enzymatic Post-digestion Labelingmentioning

confidence: 99%

Labeling Methods in Mass Spectrometry Based Quantitative Proteomics

Sap¹,

Demmers²

2012

Integrative Proteomics

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Eight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases

Cited by 242 publications

References 35 publications

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

Labeling Methods in Mass Spectrometry Based Quantitative Proteomics

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