Simultaneous Monitoring of Multiple Kinase Activities by SELDI-TOF Mass Spectrometry

Abstract

Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

“…Ciphergen Biosystems (Palo Alto, CA) developed a ProteinChip System that consists of a ProteinChip Reader (i.e., MALDI linear TOF mass spectrometer), ProteinChip Software, and related accessories that are used in conjunction with ProteinChip Arrays (Tang, Tornatore, & Weinberger, 2004). A number of reports described the use of these chips for phosphorylated peptide and protein purification (Cardone et al, 1998; Davies, 2000; Laine et al, 2000; Roig et al, 2000; Mortier et al, 2001; Stoica et al, 2001; Tassi et al, 2001; Espina et al, 2004; Liu et al, 2004; Thulasiraman et al, 2004; Bowley et al, 2005; Ge, Gibbs, & Masse, 2005; Head et al, 2005; Righetti et al, 2005; Wu, Jin, & Marsh, 2005; Le Bihan et al, 2006; Bodega et al, 2007; Akashi & Yamori, 2007; Akashi et al, 2007). One example was the use of an IMAC‐Gallium ProteinChip Array (IMAC chips were charged with a solution of 50 mM gallium nitrate) for the simultaneous analysis of a mixture of three synthetic monophosphorylated peptides, KRPpSQRHGSKY, TRDIYETDYpYRK, and KRELVEPLpTPSGEAPNQALLR, and their nonphosphorylated counterparts (Thulasiraman et al, 2004).…”

Parameter estimate of signal transduction pathways

“…The analysis of reaction mixtures by mass spectrometry methods makes the use of chromophores and radiolabelling unnecessary, since even the addition of a phosphate group to a large protein can be detected as a precise mass shift in the spectra. In vitro multiplexed assays can be performed on protein chips that are then directly analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to monitor enzyme activities [82]. Alternatively complex protein mixtures can be immobilized on micro-beads, where the enzymatic reactions can take place and be monitored by MALDI mass spectrometry [83].…”

Multiplexed G-Protein–Coupled Receptor Ca2+ Flux Assays for High-Throughput Screening

“…In addition, it can also simplify the construction and execution of target selectivity panels. Although the use of multiplexing in hit discovery has been previously reported for kinases, nuclear receptors, and GPCRs, 11,[16][17][18] there are few examples of multiplexed assay systems used for large-scale HTS and none using Ca 2+ flux assays for GPCRs. Therefore, the benefits and liabilities of multiplexed Ca 2+ flux assays to large-scale screening are unknown.…”