Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

Huttlin, Edward L.; Chen, Xiaodi; Barrett-Wilt, Gregory A.; Hegeman, Adrian D.; Halberg, Richard B.; Harms, Amy C.; Newton, Michael A.; Dove, William F.; Sussman, Michael R.

doi:10.1073/pnas.0909282106

Cited by 29 publications

(36 citation statements)

References 58 publications

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“…S1A). Therefore, any changes in phosphorylation levels caused by the addition of ABA show a reciprocal ratio when the isotope label is exchanged between experimental sets (14). This strategy is an effective quantitation platform with each reciprocal set providing a biological replicate and has been used in several laboratories.…”

Section: Resultsmentioning

confidence: 99%

“…Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used 14 N/ 15 N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5-30 min.…”

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confidence: 99%

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In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

Kline

Barrett-Wilt

Sussman

2010

Proc. Natl. Acad. Sci. U.S.A.

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216

199

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Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used 14 N/ 15 N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5-30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone.mass spectrometry | proteomics | quantitation | metabolic labeling | Arabidopsis thalianaA bscisic acid (ABA) is a phytohormone that initiates the water removal and overall dormancy program that plant embryos undergo during seed formation and germination and also mediates the cold and drought stress responses that occur in vegetative tissues (1, 2). The receptor for this hormone recently has been identified as a family of small soluble proteins known as PYR/ PYLs that are encoded by 14 genes in the Arabidopsis genome (3, 4). Conformational studies using crystallography and NMR, as well as in vitro biochemical experiments, indicate that upon ABA binding these proteins undergo a conformational change that results in an increased affinity for protein phosphatase 2C (PP2C), and this interaction results in inhibition of phosphatase activity (5-8). These results are consistent with genetic data using stable transgenic lines containing knockout and overexpression mutations and with transient expression studies of the receptors and phosphatase (9, 10).The resulting model predicts that downstream effects of ABA are mediated by early changes in the phosphoproteome induced by changes in PP2C phosphatase activity, which then can activate downstream sucrose non-fermenting 1-related subfamily 2 (SnRK2) kinases (11). To ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

Kline

Barrett-Wilt

Sussman

2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

216

199

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“…In addition to normalizing the compositions of the two diets, we used a "reciprocal labeling" strategy to control for any slight or undetected differences between the 15 N-and 14 N-labeled chows (24,25) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Quantitative assessment of the impact of the gut microbiota on lysine ε-acetylation of host proteins using gnotobiotic mice

Simón¹,

Cheng²,

Gordon³

2012

Proc. Natl. Acad. Sci. U.S.A.

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The gut microbiota influences numerous aspects of human biology. One facet that has not been thoroughly explored is its impact on the host proteome. We hypothesized that the microbiota may produce certain of its effects through covalent modification of host proteins. We focused on protein lysine ε-acetylation because of its recently discovered roles in regulation of cell metabolism, and the potential for products of microbial fermentation to interact with the lysine acetylation machinery of host cells. Germ-free mice, fed a 15 N-labeled diet for two generations, were colonized as adults with a microbiota harvested from conventionally raised mouse donors. Using high-resolution mass spectrometry, we quantified 3,891 liver and proximal colonic proteins, 558 of which contained 1,602 sites of lysine acetylation, 43% not previously described. Multiple proteins from multiple subcellular compartments underwent microbiota-associated increases in their levels of lysine acetylation at one or more residues, in one or both tissues. Acetylated proteins were enriched in functions related to energy production, respiration, and primary metabolism. A number of the acetylation events affect lysine residues at or near the active sites of enzymes, whereas others occur at locations that may affect other facets of protein function. One of these modifications, affecting Lys292 in mouse α-1-antitrypsin, was detected in the corresponding lysine of the human serum protein. Methods described in this report can be applied to other co-or posttranslational modifications, and add quantitation of protein expression and covalent modification to the arsenal of techniques for characterizing the dynamic, important interactions between gut symbionts and their hosts.gut microbiome | quantitative proteomics | spirulina | stable isotopic labeling of mammals

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“…In previous studies we have used the Apc Min/+ mouse model of familial intestinal cancer to identify proteins that are differentially expressed in tumor-bearing versus tumor-free colonic tissue and in the serum of Apc Min/+ versus Apc +/+ mice (6, 7). However, Apc Min/+ mice develop adenomas predominantly in the small intestine, not the colon, which confounds the interpretation of using the Apc Min/+ model for colon cancer studies (7–9).…”

Section: Introductionmentioning

confidence: 99%

The Concentrations of EGFR, LRG1, ITIH4, and F5 in Serum Correlate with the Number of Colonic Adenomas in ApcPirc/+ Rats

Ivancic

Irving

Jonakin

et al. 2014

Cancer Prevention Research

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The development of noninvasive methods for early detection of colon cancer is critical for the successful management of this disease. Using a targeted quantitative proteomics technique, we assessed the ability of 12 serum proteins to detect the presence of colonic polyps in the Apc Pirc/þ rat model of familial colon cancer. Serum protein candidates were selected from gene transcripts upregulated in colonic tumors of Apc Pirc/þ rats and from a prior study of serum proteins differentially expressed in mice carrying intestinal adenomas. Proteins were quantified at early stages of polyp formation in a rat cohort monitored longitudinally by colonoscopy over a period of 75 days. Of the 12 proteins monitored at three distinct time points, seven showed differential expression in at least one time point in the serum from Apc Pirc/þ rats compared with wild-type rats. Tumor multiplicity correlated with protein expression changes, and most tumors grew during the study. EGFR, LRG1, ITIH4, and F5 displayed the most robust tumor-associated protein expression changes over time. Receiver operator characteristic analysis using these four proteins resulted in a sensitivity of 100%, a specificity of 80%, and an area under the curve of 0.93 at 135 days of age, when the Pirc rats bore an average of 19 tumors in the colon and seven in the small intestine. The results of this study demonstrate that the quantitative analysis of a panel of serum proteins can detect the presence of early intestinal tumors in a rat model, and provides support for future measurements in humans. Cancer Prev Res; 7(11); 1160-9. Ó2014 AACR.

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Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

Cited by 29 publications

References 58 publications

In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

Quantitative assessment of the impact of the gut microbiota on lysine ε-acetylation of host proteins using gnotobiotic mice

The Concentrations of EGFR, LRG1, ITIH4, and F5 in Serum Correlate with the Number of Colonic Adenomas in ApcPirc/+ Rats

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