A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry

Jedrzejewski, Paul T.; Girod, Andreas; Tholey, Andreas; König, Norbert; Thullner, Sandra; Kinzel, V.; Bossemeyer, Dirk

doi:10.1002/pro.5560070227

Cited by 51 publications

(83 citation statements)

References 77 publications

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“…The selected proteins and their Brookhaven Protein Data Bank identification numbers are: rabbit aldolase 1ADO (11,18), human angiogenin 1B1I (19,20), bovine calbindin 4ICB (21,22), pig cAMP-dependent protein kinase 1CDK (23,24), horse cytochrome C 2GIW (NMR) (25,26), mouse epidermal growth factor 1EGF (NMR) (27,28), rat fatty acid-binding protein 1LFO (29,30), human fibroblast growth factor 2AFG (31,32), Aspergillus awamorii glucoamylase 3GLY (33,34), human growth hormone 1HGU (35,36), human hemoglobin 1A3N (37)(38)(39)(40)(41)(42)(43)(44)45), Escherichia coli Hpr-phosphocarrier protein 1HDN (NMR) (46,47), human hypoxanthine guanine phosphoribosyl Abbreviation: 3D, three-dimensional. ‡ To whom reprint requests should be addressed.…”

Section: Methodsmentioning

confidence: 99%

Prediction of protein deamidation rates from primary and three-dimensional structure

Robinson

2001

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

229

247

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A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and threedimensional structure in Asn deamidation is estimated.biological clocks ͉ proteins T he spontaneous deamidation of glutaminyl and asparaginyl residues causes experimentally and biologically important changes in peptide and protein structures. In asparaginyl deamidation, the primary reaction products are aspartyl and isoaspartyl. Early work on peptide and protein deamidation (1-10) established that deamidation occurs in vitro and in vivo and depends on primary sequence, three-dimensional (3D) structure, pH, temperature, ionic strength, buffer ions, and other solution properties.It was hypothesized (3, 5, 7) and then experimentally demonstrated (2, 8, 9, 11) that deamidation can serve as a biologically relevant molecular clock that regulates the timing of in vivo processes. Substantial evidence supports the hypothesis that Asn deamidation at neutral pH proceeds through a cyclic imide reaction mechanism (12)(13)(14).A procedure is needed whereby the stability of individual amides in peptides and proteins can be reliably estimated. Although it was evident to investigators 30 years ago (2-7) that protein deamidation rates depend on primary, secondary, tertiary, and quaternary protein structure, and numerous examples have been found, it was not possible to devise a useful deamidation prediction procedure until a complete library of deamidation rates as a function of primary sequence was available.A suitable library of sequence-determined Asn rates has now been published (15), and the relevance of this library has been established (16). These rates can now be combined with 3D data to provide a useful deamidation prediction procedure. Each amide residue has an intrinsic sequence-determined deamidation rate, which depends on charge distribution, steric factors, and other aspects of peptide chemistry. This primary rate is modulated by 3D structure, which usually slows the rate. In a few instances, it increases the deamidation rate.We have devised a simple procedure that is useful for predicting the relative deamidation rates of most protein Asn residues. We have tested this procedure on a complete set of all proteins for which, during a review of the literature, we found experiments specifically identifying one or more labile Asn residues in a protein and also a suitable 3...

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Section: Methodsmentioning

confidence: 99%

Prediction of protein deamidation rates from primary and three-dimensional structure

Robinson

2001

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

229

247

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“…The values of the apparent molecular mass ranged between 41.3 kDa for C 4 and 44.5 kDa for C 6 . All the mussel isoforms were slightly heavier than the bovine C-subunit used as a control (lane 2), whose molecular mass, determined by MS, was exactly 40 855.7 Da [15]. On the other hand, samples of purified mussel C 1 -C 6 were probed with both phosphoserine and phosphothreonine antibodies, and they were all serine and threonine phosphorylated, as shown in Fig.…”

Section: Characterization Of C-subunit Isoformsmentioning

confidence: 93%

“…No significant differences among C-subunit isoforms regarding the values of apparent K m for Kemptide and V max were observed. Furthermore, all the mussel isozymes were inhibited by the protein kinase inhibitor peptide [PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)] with similar I 50 values ( Table 2). The ability of mussel C-subunit isoforms to phosphorylate proteins in vitro was also investigated.…”

Section: Kinetic Characterization Of C-subunit Isoforms and Protein Pmentioning

confidence: 99%

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Identification of multiple isoforms of the cAMP‐dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis

2008

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Several isoforms of the cAMP‐dependent protein kinase catalytic subunit (C‐subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue‐specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C‐subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C‐subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Cα‐isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C‐subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C‐subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

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“…Several reports have been published which highlight the advantages of capillary LC with ESI-MS in, for example, the field of DNA-adduct analysis [10][11][12] and peptide analysis. [13][14][15] The advantages of capillary LC coupled to ESI-MS could prove to be of benefit in many analytical areas, including the characterisation of metabolites within complex biological fluids. The sensitivity gain will clearly be of use for identification of low level metabolites (particularly circulating metabolites), which is increasingly likely as drug efficacy is increased and dosage subsequently decreased.…”

mentioning

confidence: 99%

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Dear¹,

Ayrton²,

Plumb³

et al. 1999

Rapid Commun. Mass Spectrom.

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Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.

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A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry

Cited by 51 publications

References 77 publications

Prediction of protein deamidation rates from primary and three-dimensional structure

Prediction of protein deamidation rates from primary and three-dimensional structure

Identification of multiple isoforms of the cAMP‐dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

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