The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Zeller, Martin; König, Simone

doi:10.1007/s00216-003-2391-2

Cited by 28 publications

(17 citation statements)

References 206 publications

(272 reference statements)

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“…Surprisingly, two of these phosphorylation sites (Tyr 163 and Ser 171 ) were not identified by mass spectrometry, possibly because MELK was phosphorylated substoichiometrically on these sites. Other groups have also noted that the detection of all phosphorylation sites in a protein remains a most challenging analytical task, even when sophisticated techniques of mass spectrometry are adopted (29,30). In any case, our findings nicely illustrate the importance of using multiple, independent approaches for the mapping of phosphorylation sites.…”

Section: Discussionsupporting

confidence: 67%

Substrate Specificity and Activity Regulation of Protein Kinase MELK

Beullens

Vancauwenbergh

Morrice

et al. 2005

Journal of Biological Chemistry

100

109

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Maternal embryonic leucine zipper kinase (MELK) is a proteinSer/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr 167 The AMP-activated protein kinase (AMPK) 5 is the best characterized member of the subfamily of AMPK-related protein Ser/Thr kinases (1, 2). It is allosterically activated by AMP and controls processes that restore the energy charge in the cell. The other members of the subfamily of AMPK-related kinases include the protein kinases ARK5, BRSK, MARK, MELK, QIK, NUAK, and SNARK, which fulfill functions in processes as diverse as cell cycle progression, cell survival, cell differentiation, and microtubule stability (3-6). With the exception of maternal embryonic leucine zipper kinase (MELK), which is activated by autophosphorylation, the AMPK-related kinases are activated through phosphorylation of their T-loop by protein kinase LKB1 (5, 7) and the calcium-and calmodulin-dependent protein kinase II (8).The catalytic domain of the AMPK-related protein kinases is located in the N terminus of their catalytic subunit. Some of the AMPK-related protein kinases, including MELK, have an ubiquitin-associated (UBA) domain adjacent to the catalytic domain and a C-terminal kinase-associated 1 (KA1) domain. The function of the latter domains are poorly understood. UBA domains are known to bind (poly)ubiquitin and have been suggested to thereby prevent additional ubiquitination and proteosomal degradation of the target protein (9 -12). In some proteins, UBA domains appear to function as dimerization domains (13). Between the UBA and the KA1 domains, MELK contains a TP dipeptide-rich domain that is phosphorylated in mitotically arrested cells and mediates binding to the transcription and splicing factor NIPP1 (14).Preliminary evidence implicates MELK in various cellular processes. MELK binds tightly to the zinc finger-like protein ZPR9 and causes its nuclear accumulation (15, 16). In the nucleus, ZPR9 itself interacts with the transcription factor B-Myb, a regulator of cell proliferation and differentiation, and enhances its transcriptional activity. Another interactor of MELK is the transcription and splicing factor NIPP1, but the binding of NIPP1 requires the phosphorylation of MELK on a specific threonine in its TP dipeptide-rich domain (14). Because wild-type MELK, but not a NIPP1-binding mutant, is a potent inhibitor of pre-mRNA splicing in nuclear extracts and because the MELK-NIPP1 interaction is increased during mitosis, it has been proposed that MELK contributes to the ending of pre-mRNA splicing just before mitosis (14). A third protein ligand of MELK is Cdc25B, a protein-tyrosine phosphata...

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Section: Discussionsupporting

confidence: 67%

Substrate Specificity and Activity Regulation of Protein Kinase MELK

Beullens

Vancauwenbergh

Morrice

et al. 2005

Journal of Biological Chemistry

100

109

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“…These considerations demostrate how mass spectrometry in the field of phosphorylation mapping is still not a routine activity in most laboratories. [17][18][19] One of the obvious drawbacks of our heteronuclear NMR methodology is the need to obtain the assignment of the phosphorylated residues. After identification of these residues as serines, based on the CR/Cβ chemical shifts extracted from a CBCANH spectrum, our initial assignment was only based on the two residues surrounding the phospho-Ser.…”

Section: Discussionmentioning

confidence: 99%

“…Even when the enzymatic reaction is executed in a test tube with a well-known protein substrate and recombinant kinase, current analytical methods still face serious shortcomings. Despite impressive methodological developments, the necessity to preprocess the sample, the tendency of phosphorylation sites on Tau to cluster, and the technical difficulties inherently associated with the detection and quantification of the highly charged phospho-peptide prevent mass spectrometry from being a routine analytical tool to establish a precise phosphorylation pattern. − Immunodetection by antibodies is equally limited by the requirement of a comprehensive antibody library against all possible epitopes and by the absence of a fully phosphorylated standard for every combination of sites in order to quantify the level of phosphorylation. New analytical methods able to rapidly identify the phosphorylation sites for a given kinase or combination of kinases and quantify their extent of phosphate incorporation are still needed.…”

Section: Introductionmentioning

confidence: 99%

NMR Analysis of a Tau Phosphorylation Pattern

et al. 2006

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The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimer's diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

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“…In the triple quadrupole mass spectrometer, ions of the selected target molecules among the ions generated by ESI are extracted by the first quadrupole (Q1) [101]. The ions extracted by the Q1 are collided with Ar gas and fragmented by the second quadrupole (Q2) [102,103]. The fragmentation method is called collision-induced dissociation (CID).…”

Section: Lc/ms/msmentioning

confidence: 99%

Rapidity and Precision of Steroid Hormone Measurement

Osaka

2022

JCM

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Steroids are present in all animals and plants, from mammals to prokaryotes. In the medical field, steroids are commonly classified as glucocorticoids, mineralocorticoids, and gonadal steroid hormones. Monitoring of hormones is useful in clinical and research fields for the assessment of physiological changes associated with aging, disease risk, and the diagnostic and therapeutic effects of various diseases. Since the discovery and isolation of steroid hormones, measurement methods for steroid hormones in biological samples have advanced substantially. Although immunoassays (IAs) are widely used in daily practice, mass spectrometry (MS)-based methods have been reported to be more specific. Steroid hormone measurement based on MS is desirable in clinical practice; however, there are several drawbacks, including the purchase and maintenance costs of the MS instrument and the need for specialized training of technicians. In this review, we discuss IA- and MS-based methods currently in use and briefly present the history of steroid hormone measurement. In addition, we describe recent advances in IA- and MS-based methods and future applications and considerations.

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The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Cited by 28 publications

References 206 publications

Substrate Specificity and Activity Regulation of Protein Kinase MELK

Substrate Specificity and Activity Regulation of Protein Kinase MELK

NMR Analysis of a Tau Phosphorylation Pattern

Rapidity and Precision of Steroid Hormone Measurement

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