Phospho-proteomics: Evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis

Larsen, Martin R.; Sørensen, Grith Lykke; Fey, Stephen J.; Larsen, Peter Mose; Roepstorff, Peter

doi:10.1002/1615-9861(200102)1:2<223::aid-prot223>3.3.co;2-2

Cited by 67 publications

(89 citation statements)

References 5 publications

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“…5, D and E). Phosphatase alkaline (Roche) experiments were performed as described (60), on the same spot directly onto the MALDI target plate with ␣-cyano-4-hydroxycinnamic acid matrix. The characterization of dephosphorylation of peptides was performed with BioTools software (Bruker) by observing the loss of 80 Da of mass of the phosphopeptides after alkaline phosphatase treatment.…”

Section: Methodsmentioning

confidence: 99%

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

Bouillet¹,

Genest²,

Jourlin-Castelli³

et al. 2016

Journal of Biological Chemistry

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Edited by Thomas SöllnerHere, we show that a partner-switching system of the aquatic Proteobacterium Shewanella oneidensis regulates post-translationally S (also called RpoS), the general stress response sigma factor. Genes SO2118 and SO2119 encode CrsA and CrsR, respectively. CrsR is a three-domain protein comprising a receiver, a phosphatase, and a kinase/anti-sigma domains, and CrsA is an anti-sigma antagonist. In vitro, CrsR sequesters S and possesses kinase and phosphatase activities toward CrsA. In turn, dephosphorylated CrsA binds the anti-sigma domain of CrsR to allow the release of S . This study reveals a novel pathway that post-translationally regulates the general stress response sigma factor differently than what was described for other proteobacteria like Escherichia coli. We argue that this pathway allows probably a rapid bacterial adaptation.

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Section: Methodsmentioning

confidence: 99%

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

Bouillet¹,

Genest²,

Jourlin-Castelli³

et al. 2016

Journal of Biological Chemistry

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“…Calf intestine alkaline phosphatase (1 l, New England Biolabs) in 50 mM NH 4 CO 3 , pH 8.0, was added on top of the dried analyte/matrix MALDI spot for 1 h at 37°C as described in Ref. 22. After incubation, 1 l of matrix was added to recrystallize the sample.…”

Section: Methodsmentioning

confidence: 99%

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Schilling

Gafni

Torcassi

et al. 2006

Journal of Biological Chemistry

135

114

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Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing fulllength myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt.

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“…Since the peptide was known to be phosphorylated, DHB was used as the matrix because it does not promote loss of H 3 PO 4 as readily as HCCA does [44].…”

Section: Application Of Pseudo-ms 3 To the Localization Of A Phosphormentioning

confidence: 99%

Pseudo-MS³ in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Raska

Parker

Huang

et al. 2002

J. Am. Soc. Mass Spectrom.

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Both the matrix selected and the laser fluence play important roles in MALDI-quadrupole/ time of flight (QqTOF) fragmentation processes. "Hot" matrices, such as ␣-cyano-4-hydroxycinnamic acid (HCCA), can increase fragmentation in MS spectra. Higher laser fluence also increases fragmentation. Typical peptide fragment ions observed in the QqTOF are a, b, and y ion series, which resemble low-energy CID product ions. This fragmentation may occur in the high-pressure region before the first mass-analyzing quadrupole. Fragment ions can be selected by the first quadrupole (Q1), and further sequenced by conventional MS/MS. This allows pseudo-MS 3 experiments to be performed.For peptides of higher molecular weight, pseudo-MS 3 can extend the mass range beyond what is usually accessible for sequencing, by allowing one to sequence a fragment ion of lower molecular weight instead of the full-length peptide. Peptides that predominantly show a single product ion after MS/MS yield improved sequence information when this technique is applied. This method was applied to the analysis of an in vitro phosphorylated peptide, where the intact enzymatically-generated peptide showed poor dissociation via MS/MS. Sequencing a fragment ion from the phosphopeptide enabled the phosphorylation site to be unambiguously determined. A fter the matrix-assisted laser desorption/ionization (MALDI) technique was first developed [1][2][3][4][5], there were extensive studies on the selection of matrices optimized for various types of lasers [6 -9]. Although most researchers are now primarily using 337 nm nitrogen lasers and only a few selected matrices, there has been much discussion of MALDI matrices with regard to mechanisms of ion formation [10 -24].In-source decay (ISD [25,26]) and post-source decay (PSD [27]) in MALDI/TOF-MS have been studied for the purpose of determining the mechanism of MALDI ionization, and have led to the classification of matrices such as ␣-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB) as "hot" or "cold" based originally on the amount of glycoprotein fragmentation they induce [28].The differences in fragmentation observed with various matrices have been attributed to differences in initial velocities of the ions generated [29 -31], with higher initial velocities leading to cooling and less fragmentation. It has been noted that, for a given matrix, all analyte ions below 25 kDa appear to have the same initial velocity irrespective of mass, but the initial velocity depends on the matrix [31]. An early hypothesis that the internal energy and velocity of both analyte and matrix are a function of matrix sublimation temperatures has found empirical support [32]. Zenobi and co-workers found, however, that the internal energy distributions of analyte ions did not correlate with matrix sublimation temperatures, but instead depended on gas-phase proton transfer reactions [20,21,23]. Increasing laser fluence has been shown to increase analyte ion dissociation via in-source decay (ISD) [33][34][35][36][37][38...

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Phospho-proteomics: Evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis

Cited by 67 publications

References 5 publications

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Pseudo-MS³ in a MALDI orthogonal quadrupole-time of flight mass spectrometer

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Phospho-proteomics: Evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis

Cited by 67 publications

References 5 publications

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

The General Stress Response σS Is Regulated by a Partner Switch in the Gram-negative Bacterium Shewanella oneidensis

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer

Contact Info

Product

Resources

About

Pseudo-MS³ in a MALDI orthogonal quadrupole-time of flight mass spectrometer