Profiling Kinase Activities by Using a Peptide Chip and Mass Spectrometry

Min, Dal‐Hee; Jing, Su; Mrksich, Milan

doi:10.1002/ange.200461061

Cited by 51 publications

(33 citation statements)

References 38 publications

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“…The anti-phosphoprotein antibody can be used in many analytical procedures such as the enzymelinked immunosorbent assay, Western blotting, immunocytochemistry, and immunoprecipitation. Recently a few high throughput methods for defining a number of phosphorylation events were developed using a peptide chip followed by MS (8) and surface plasmon resonance imaging (9). Chemical labeling of the phosphate group has also been used for phosphospecific site mapping in peptide mass fingerprinting and subsequent MS analysis (10 -13).…”

mentioning

confidence: 99%

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Kinoshita–Kikuta

Aoki

Kinoshita

et al. 2007

Molecular & Cellular Proteomics

133

113

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Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn 2؉ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3␤, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly upshifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC 50 value of 1.6 M in the presence of 0.10 mM ATP. Molecular & Cellular Proteomics 6:356 -366, 2007.Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1, 2). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4). T...

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mentioning

confidence: 99%

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Kinoshita–Kikuta

Aoki

Kinoshita

et al. 2007

Molecular & Cellular Proteomics

133

113

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“…[52] Moreover, proof-of-concept examples have been described for detecting kinase-mediated peptide phosphorylation on microarrays by using surface plasmon resonance [31] or MALDI-TOF mass spectrometry. [7] 3. Applications…”

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confidence: 99%

“…[5][6][7] SPOT synthesis is technically very simple and flexible and does not require any expensive laboratory automation or synthesis hardware. The resulting spot size is defined by the dispensed volume as well as the physical properties of the surface used.…”

Section: Array Production and Assay Principlesmentioning

confidence: 99%

Peptide Arrays for Kinase Profiling

Schutkowski

Reineke²,

Reimer³

2005

ChemBioChem

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“…[7] Mrksich and co-workers applied this principle to characterize SAMs, and further applied it as a signal readout tool in SAM-based biochips engineered for enzyme assays, protein-protein interactions, and chemical screening. [8] In this current study, SAMs on a gold substrate were selectively desorbed and removed at designated regions by MALDI-TOF MS, and the exposed bare gold regions were then backfilled with a second alkanethiol. [9] For the patterning of ligands, we first selectively desorbed and removed SAMs with tri(ethyleneglycol) (EG 3 ) groups at designated regions on a gold substrate by using a MALDI-TOF MS nitrogen laser.…”

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confidence: 99%

Mass Spectrometry Assisted Lithography for the Patterning of Cell Adhesion Ligands on Self‐Assembled Monolayers

et al. 2009

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Pattern of events: A simple and flexible method has been developed for patterning cell adhesion ligands. Locally erasing self-assembled monolayers with tri(ethyleneglycol) groups on a gold substrate by using a MALDI-TOF MS nitrogen laser and filling the exposed gold surface with an alkanethiol presenting carboxylic acid groups enables subsequent immobilization of maleimide and a cell adhesion peptide, which can then recognize cells (see scheme).

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Profiling Kinase Activities by Using a Peptide Chip and Mass Spectrometry

Cited by 51 publications

References 38 publications

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Peptide Arrays for Kinase Profiling

Mass Spectrometry Assisted Lithography for the Patterning of Cell Adhesion Ligands on Self‐Assembled Monolayers

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