Identification of Phosphorylation Sites in Proteins Separated by Polyacrylamide Gel Electrophoresis

Zhang, Xiaolong; Herring, Christopher J.; Romano, Patrick R.; Szczepanowska, Joanna; Brzeska, Hanna; Hinnebusch, and Alan G.; Qin, Jun

doi:10.1021/ac971207m

Cited by 181 publications

(173 citation statements)

References 41 publications

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“…Peptides can therefore be detected by MS/MS that would otherwise be lost in the background noise of full mass range MS. This property of ion traps has been exploited, for example, to obtain MS/MS data on peptides initially detected by matrix-assisted laser desorption-ionization-time-offlight MS (20,21). To take advantage of the sensitivity of the ion trap for MS/MS new ICAT reagents were synthesized that incorporate three (rather than eight) deuterium atoms in the heavy version (Fig.…”

Section: Resultsmentioning

confidence: 99%

Selective Detection of Membrane Proteins Without Antibodies

Arnott¹,

Kishiyama²,

Luis³

et al. 2002

Molecular & Cellular Proteomics

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A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude. Molecular & Cellular Proteomics 1:148 -156, 2002.

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Section: Resultsmentioning

confidence: 99%

Selective Detection of Membrane Proteins Without Antibodies

Arnott¹,

Kishiyama²,

Luis³

et al. 2002

Molecular & Cellular Proteomics

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“…Mass Spectrometric Analysis-After SDS-PAGE of the purified V 1 /V o ATPase, the 100-kDa and the 17-kDa Coomassie-stained bands, respectively, were prepared for mass spectrometry in a procedure adapted from Shevchenko et al (17) and Zhang et al (18). Briefly, the proteins were digested by trypsin or chymotrypsin in the gel, and the peptides were mapped using MALDI-MS (TofSpec-2E, Micromass Ltd., Manchester, UK).…”

Section: Methodsmentioning

confidence: 99%

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Huss

Ingenhorst

König

et al. 2002

Journal of Biological Chemistry

254

244

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“…The observed band was excised from the membrane for Nterminal sequencing by Edman degradation in the Baylor College of Medicine protein chemistry core. Proteins immunoprecipitated by anti-RGS9-1c IgG coupled Sepharose beads were eluted by 0.1 M glycine, pH 3.0, separated by SDS͞PAGE, and subjected to in-gel trypsin digestion followed by a combination of matrix-assisted laser desorption͞ionization time-of-f light mass spectrometry and on-line capillary liquid chromatography electrospray tandem ion trap mass spectrometry as described (37).…”

Section: Methodsmentioning

confidence: 99%

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Wensel²

2002

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

157

150

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The regulator of G protein signaling (RGS)-9-1⅐G␤5 complex forms the GTPase accelerating protein for G␣t in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G␤5, G␣t, and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.T o ensure high efficiency of phototransduction, the essential components of the phototransduction cascade are packed at a high density on photoreceptor outer segment disk membranes (1), where phototransduction occurs. One challenge in photoreceptor biology has been to determine the mechanisms by which these molecules and their modulators are localized to the disk membranes. This question has been particularly puzzling for regulator of G protein signaling (RGS)-9-1, which is the GTPase accelerating protein (GAP) for the visual G protein G ␣t (2-4).RGS9-1 plays an essential role in setting the timing of the recovery phase of light responses in vertebrate photoreceptors (5, 6). It is a tightly bound peripheral membrane protein (7), but has no obvious features that target it to membranes and is largely soluble when expressed in vitro (8, 9). RGS9-1 forms a constitutive complex with its obligate subunit, G ␤5 (5, 8, 10), and it also interacts with the inhibitory subunit of the effector for G ␣t , cGMP phosphodiesterase-␥ (PDE␥) (11)(12)(13)(14). Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2, 9, 15). Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16-18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).We describe here the identification by coimmunoprecipitation of a heterotetrameric complex containing RGS9-1, G ␤5L , G ␣t , and a previously unknown photoreceptor-specific protein, R9AP. R9AP interacts with the N-terminal domain of RGS9-1 and has a transmembrane helix at its C terminus, allowing it to serve as the RGS9-1-anchor protein. Materials and MethodsBuffers. The following standard buffers were used: low-salt buffer (5 mM Tris⅐HCl͞0.5 mM MgCl 2 ), GAPN buffer (10 mM Hepes͞ 100 mM NaCl͞2 mM MgCl 2 ), high-salt buffer (10 mM Hepes͞1 M NH 4 Cl͞2 mM MgCl 2 ), urea buffer (4 M urea͞5 mM Tris⅐HCl). The pH of all buffers was adjusted to 7.4-7.5, and 1 mM DTT and Ϸ20 mg/liter solid PMSF were added before use.P...

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Identification of Phosphorylation Sites in Proteins Separated by Polyacrylamide Gel Electrophoresis

Cited by 181 publications

References 41 publications

Selective Detection of Membrane Proteins Without Antibodies

Selective Detection of Membrane Proteins Without Antibodies

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

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