Quantitative determination of phosphoserine by high-performance liquid chromatography as the phenylthiocarbamyl-s-ethylcysteine

Meyer, Helmut E.; Swiderek, Kristine M.; Hoffmann-Posorske, Edeltraut; Korte, H.; Heilmeyer, Ludwig

doi:10.1016/s0021-9673(01)84994-3

Cited by 35 publications

(12 citation statements)

References 13 publications

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“…The actual phosphorylation was proved chemically dephosphorylating the peptide. 39,40 The sample was treated with 10 ml of Ba(OH) 2 for 30 min at room temperature. The reaction was quenched by adding 2 ml of formic acid and the sample was desalted using ZipTip (Millipore).…”

Section: Identification Of Ck2 Phosphorylation Site In Akt1mentioning

confidence: 99%

Protein kinase CK2 phosphorylates and upregulates Akt/PKB

et al. 2005

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Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the antiapoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.

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Section: Identification Of Ck2 Phosphorylation Site In Akt1mentioning

confidence: 99%

Protein kinase CK2 phosphorylates and upregulates Akt/PKB

et al. 2005

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“…It was therefore initially surprising to find that the serine at position 3 of T2, and not the Nterminal serine, was the residue converted to Sethylcysteine (table 1). However, Meyer et al [16] have reported that N-terminal (or C-terminal) phosphoserine residues do not convert to Sethylcysteine following incubation with ethanethiol in NaOH. The presence of S-ethylcysteine at position 3 implies that this residue is esterified (presumably to phosphate) in the purified peptide.…”

Section: Purification and Analysis Of Hsl Phosphopeptidesmentioning

confidence: 99%

Primary structure of the site on bovine hormone‐sensitive lipase phosphorylated by cyclic AMP‐dependent protein kinase

et al. 1988

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The primary structure of a region on hormone‐sensitive lipase was determined to be: Lys‐Thr‐Glu‐Pro‐Met‐Arg‐Arg‐Ser‐Val‐Ser‐Glu‐Ala‐Ala‐Leu‐Thr‐Gln‐Pro‐Glu‐Gly‐Pro‐Leu‐Gly‐Thr‐Asp‐Ser‐Leu‐Lys. Ser‐8 was the only residue in the intact protein phosphorylated by cyclic AMP‐dependent protein kinase. However, Ser‐10 also appeared to be present in a phosphorylated form, suggesting that it is a target for a distinct protein kinase in vivo.

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“…A further enrichment approach is based on the fact that phosphoserines and phosphothreonines are not stable in strong alkaline conditions [17,18,19,20]. The phosphate ester is cleaved in the presence of strong bases, such as NaOH or Ba(OH) 2 , generating dehydroalanine species which can react with different nucleophiles, such as thiols, amines or alcohols.…”

Section: Introductionmentioning

confidence: 99%

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry

et al. 2003

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beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for beta-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available alpha-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.

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Quantitative determination of phosphoserine by high-performance liquid chromatography as the phenylthiocarbamyl-s-ethylcysteine

Cited by 35 publications

References 13 publications

Protein kinase CK2 phosphorylates and upregulates Akt/PKB

Protein kinase CK2 phosphorylates and upregulates Akt/PKB

Primary structure of the site on bovine hormone‐sensitive lipase phosphorylated by cyclic AMP‐dependent protein kinase

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry

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