Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry

Tao, W. Andy; Wollscheid, Bernd; O’Brien, Robert V.; Eng, Jimmy K.; Li, Xiaojun; Bodenmiller, Bernd; Watts, Julian D.; Hood, Leroy; Aebersold, Ruedi

doi:10.1038/nmeth776

Cited by 296 publications

(252 citation statements)

References 45 publications

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“…Firstly, the carboxyl groups on the peptides are O-methyl-esterified to prevent them from interacting in subsequent steps and causing nonspecific purification. Phosphopeptides are derivatized by a sulphydryl group and subsequently linked to iodoacetyl groups immobilized on a synthetic polymer solid support [102] or glass beads [103] through phosphoamidate chemistry. The phosphate groups are reconstituted by acid hydrolysis of the phosphoramidate bonds (e.g., using TFA [101]), facilitating the identification of the phosphorylation sites by MS.…”

Section: Chemical Derivatization Strategiesmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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Section: Chemical Derivatization Strategiesmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

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“…After incubation at 48C for 2 h, the beads were washed three times each with 500 mL of lysis buffer, 50 mM TMAB (1 M trimethylammonium bicarbonate; Fluka), and sterile distilled water. Proteins binding to the beads were eluted with 150 mL of 50 mM TMAB containing 0.1% Rapigest and digested with trypsin as described (Tao et al, 2005;Zhou et al, 2007). Tryptic peptides were analyzed by nanoflow liquid chromatography-tandem mass spectrometry on a high-resolution hybrid linear ion trap orbitrap mass spectrometer (LTQ-Orbitrap XL; ThermoFisher) coupled to an Agilent Nanoflow LC system.…”

Section: Affinity Purification and Mass Spectrometry Analysismentioning

confidence: 99%

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

et al. 2010

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Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin b-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

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“…17 Many such efforts involve combining two or more enrichment steps to ensure selective isolation of phosphopeptides. 7,18 In this study we introduce a new approach to specific phosphopeptide detection that relies on the metal coordination properties of the phosphate group, but is independent of the amino acid residue that is phosphorylated and does not require LC separation of the peptide mixture. We call this method Metal Affinity Capture Tandem Mass Spectrometry, or MAC-MSMS for short.…”

Section: Introductionmentioning

confidence: 99%

Metal Affinity Capture Tandem Mass Spectrometry for the Selective Detection of Phosphopeptides

2006

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We report a new method called Metal Affinity Capture that when coupled with tandem mass spectromery (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga III or Fe III and N α ,N α -bis-(carboxymethyl)lysine (LysNTA) in solution, and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H) + ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH < 5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69 Ga-71 Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low nanomolar protein concentrations.

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Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry

Cited by 296 publications

References 45 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

Metal Affinity Capture Tandem Mass Spectrometry for the Selective Detection of Phosphopeptides

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