Selective Detection of Membrane Proteins Without Antibodies

Arnott, David; Kishiyama, Adrianne; Luis, Elizabeth; Ludlum, Sarah G.; Marsters, James C.; Stults, John T.

doi:10.1074/mcp.m100027-mcp200

Cited by 79 publications

(84 citation statements)

References 42 publications

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“…It is of interest to compare the present approach for testing hypotheses with alternatives that use LC-MS͞MS (ref. 50 and references cited therein) where the number of hypotheses that can be tested is limited by the chromatographic peak width and the time required to acquire an MS͞MS spectrum. For example, a combination of isotope-coded affinity tag (ICAT) methodology and online HPLC-MS͞MS was recently used to test for the presence of a protein of interest from a complex mixture of proteins.…”

Section: Discussionmentioning

confidence: 99%

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Kalkum

Lyon

Chait

2003

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

105

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A method is presented for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum-sensing peptides. The procedure, based on targeted multistage MS, uses a novel matrix-assisted laser desorption͞ionization-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, and chemical noise) that impair observation of low abundance peptides from complex biological matrixes. Here, secreted peptides that are hypothesized to be present in the supernatant, but that may not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multistage MS. Highly specific fragmentation signatures enable unambiguous identification of the peptides of interest and differentiation of the signals from the background. As examples, we demonstrate the rapid (<1 min) determination of the mating type of cells in colonies of Saccharomyces cerevisiae and the elucidation of autoinducing peptides (AIPs) from supernatants of pathogenic Staphylococci. We confirm the primary structures of the agrD encoded cyclic AIPs of Staphylococcus aureus for groups I, II, and IV and provide direct evidence that the native group-III AIP is a heptapeptide (INCDFLL). We also show that the homologous peptide from Staphylococcus intermedius is a nonapeptide (RIPTSTGFF) with a lactone ring formed through condensation of the serine side chain with the C terminus of the peptide. This is the first demonstration of cyclization in a staphylococcal AIP that occurs via lactone formation. These examples demonstrate the analytical power of the present procedure for characterizing secreted peptides and its potential utility for identifying microorganisms.A ssays that provide information on specific peptides that are secreted by living cells (e.g., toxins, pheromones, virulence factors, and quorum-sensing peptides) can be valuable as diagnostic aids. For example, the presence and identity of a specific microorganism may be inferred by detection of characteristic secreted peptides, which may also be used to distinguish between subtypes of a given strain. Several authors have described the use of MS to identify microorganisms based on fingerprint masses of protein constituents as well as secreted peptides and proteins (1). Recently, electrospray ionization-Fourier transform tandem MS (MS͞MS) of intact proteins has been applied for the characterization of biomarkers from Bacillus cereus T spores (2), and a combined approach of single-stage MS and MS͞MS was used to identify proteins secreted from adipocytes (3). Because these methods rely on the initial detection by single-stage MS of intact peptide ions from a highly complex milieu, it may be difficult to detect trace amounts of the secreted peptides. In addition to limitations imposed by dynamic range and signal suppression effects, low-level signals of interest tend to disappear into the ''chemical noise '' (4-6). An approach that has been widely...

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Section: Discussionmentioning

confidence: 99%

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Kalkum

Lyon

Chait

2003

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

105

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“…Second, 18 O/ 16 O-ratios could also be calculated from MS/MS spectra [22] because y-ions retain the C-terminally labeled lysine or arginine residues. However, special care needs to be taken when using these ions.…”

Section: Protein Quantification By Lc-esi-ms/ms On Ion Trapmentioning

confidence: 99%

Trypsin catalyzed ¹⁶O-to-¹⁸O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

Heller¹,

Mattou²,

Menzel³

et al. 2003

J. Am. Soc. Mass Spectrom.

144

186

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Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, 13 C, 18 O, or 15 N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease 16 O-to-18 O catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every proteasederived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two 18 O isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESIand MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of 16 O/ 18 O-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins. (J Am Soc Mass Spectrom 2003, 14, 704 -718)

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“…Tandem mass spectrometry was performed on a Q-STAR QTOF with a MALDI source (Applied Biosystems). For additional sequence confirmation, tryptic peptides were analyzed with capillary reverse phase high performance liquid chromatography in-line with an electrospray ionization LCQ Deca ion-trap mass spectrometer (Thermo Finnigan) using a guided single ion monitoring approach as previously described (25,26).…”

Section: Methodsmentioning

confidence: 99%

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Nojiri

Loyet²,

Klenchin

et al. 2009

Journal of Biological Chemistry

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CAPS (Ca 2؉ -dependent activator protein for secretion) functions in priming Ca 2؉ -dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca 2؉ -dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca 2؉ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca 2ϩ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca 2ϩ -dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1 2 (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca 2ϩ -triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2-4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12)(13)(14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15,16). Several SNARE...

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Selective Detection of Membrane Proteins Without Antibodies

Cited by 79 publications

References 42 publications

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Trypsin catalyzed ¹⁶O-to-¹⁸O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

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Selective Detection of Membrane Proteins Without Antibodies

Cited by 79 publications

References 42 publications

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

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Trypsin catalyzed ¹⁶O-to-¹⁸O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers