Mass-tag technology responding to intracellular signals as a novel assay system for the diagnosis of tumor

Abstract

A novel mass spectrometry-based assay system for determining protein kinase activity employing mass-tagged substrate peptide probes was used for the diagnosis of tumors. Two peptide probes (H-type and D-type) were synthesized containing the same substrate peptide sequence for protein kinase C (PKC). The molecular weights of the two probes differ because of the incorporation of deuterium into the acetyl groups of the D-type probe. The lysates of the normal and tumor tissue were prepared and reacted with the H-and D-type peptide probes, respectively. The PKC activities of the normal and tumor tissues can be compared simply and directly by calculating the phosphorylated ratio to each peptide probe, obtained from the peak intensity of the mass spectrum after mixing of the two reaction solutions. The phosphorylation ratio for the reaction of the H-type peptide probe with the tumor tissue lysate (B16 melanoma) was more than three times higher than that of the D type peptide probe with the normal skin tissue lysate. These results show that the novel assay system for detecting protein kinase activity using mass-tag technology can be a simple and useful means to profile protein kinase activity for cell or tissue lysate samples, and can be applied to the diagnosis of tumors. (J Am Soc Mass Spectrom 2007, 18, 106 -112)

Effect of the addition of diammonium citrate to α-cyano-4-hydroxycinnamic acid (CHCA) matrix for the detection of phosphorylated peptide in phosphorylation reactions using cell and tissue lysates

“…In a previous study, we found that the substrate (FKKQGSFAKKK) showed a higher phosphorylation ratio on PKC␣ [22]. Here, the same substrate was used in the phosphorylation reaction for PKC␣.…”

“…Means of the phosphorylation ratio obtained from three spots selected manually on the crystals were used as results for assay purposes. The phosphorylation ratio is defined as the ratio of ion intensities for the phosphorylated to unphosphorylated material, and this was calculated as described previously [22] .…”

Technologies for the Use of Protein Kinases into Medical Applications

“…Fluorescence polarization, bead techniques using fluorimetry and colorimetric assays offer a way to reach this goal. Mass spectrometry is also a promising approach Kang et al, 2007;Shigaki et al, 2006). Peptide arrays are another promising technology for detail evaluation of cellular conditions.…”

Role of estrogenic compounds (diethylstibestrol, 17β‐estradiol, and bisphenol A) in the phosphorylation of substrate by protein kinase Cα

“…In the present study, CHCA was used as a matrix for analysis. Phosphorylation of the substrate by PKCα was identified by an increase in the m/z value of +80 Da ( Figure 1) [18,19].…”