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Miao Liu,
Jinxuan Hou,
Lin Huang,
Xin Huang, Tyler H Heibeck,
Rui Zhao,
Ljiljana Pasa-Tolic,
Richard D Smith,
Yan Li,
Kai Fu,
Zhixin Zhang,
Steven H Hinrichs,
Shi-Jian Ding
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ABSTRACT: Here we present a novel and robust method for the identification of protein S-nitrosylation sites in complex protein mixtures. The approach utilizes the cysteinyl affinity resin to selectively enrich S-nitrosylated peptides reduced by ascorbate followed by nanoscale liquid chromatography tandem mass spectrometry. Two alkylation agents with different added masses were employed to differentiate the S-nitrosylation sites from the non-S-nitrosylation sites. We applied this approach to MDA-MB-231 cells treated with Angeli's salt, a nitric oxide donor that has been shown to inhibit breast tumor growth and angiogenesis. A total of 162 S-nitrosylation sites were identified and an S-nitrosylation motif was revealed in our study. The 162 sites are significantly more than the number reported by previous methods, demonstrating the efficiency of our approach. Our approach will further facilitate the functional study of protein S-nitrosylation in cellular processes and may reveal new therapeutic targets.
Analytical Chemistry 09/2010; 82(17):7160-8. · 5.86 Impact Factor
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Xu Zhang,
Matthew E Monroe,
Baowei Chen,
Mark H Chin, Tyler H Heibeck,
Athena A Schepmoes,
Feng Yang,
Brianne O Petritis,
David G Camp,
Joel G Pounds,
Jon M Jacobs,
Desmond J Smith,
Diana J Bigelow,
Richard D Smith,
Wei-Jun Qian
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ABSTRACT: Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.
Molecular & Cellular Proteomics 06/2010; 9(6):1199-208. · 7.40 Impact Factor
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Tyler H Heibeck,
Shi-Jian Ding,
Lee K Opresko,
Rui Zhao,
Athena A Schepmoes,
Feng Yang,
Aleksey V Tolmachev,
Matthew E Monroe,
David G Camp,
Richard D Smith,
H Steven Wiley,
Wei-Jun Qian
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ABSTRACT: Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here, we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying antiphosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that a majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.
Journal of Proteome Research 07/2009; 8(8):3852-61. · 5.11 Impact Factor
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Daniel López-Ferrer, Tyler H Heibeck,
Konstantinos Petritis,
Kim K Hixson,
Weijun Qian,
Matthew E Monroe,
Anoop Mayampurath,
Ronald J Moore,
Mikhail E Belov,
David G Camp,
Richard D Smith
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ABSTRACT: A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with (18)O was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired byproducts or modifications. When this novel workflow was used, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and (18)O-labeled in <10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.
Journal of Proteome Research 09/2008; 7(9):3860-7. · 5.11 Impact Factor
