Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics

Abstract

To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Recent advances in blood‐related proteomics

“…5C), and was one of the proteins apparently down-regulated in monocytes of patients presenting with rheumatoid arthritis [145]. Swiprosin 1 has been recently shown to be implicated in phosphotyrosinebased signaling events involved in the cellular stimulation of early growth factors [164]. In their study, the authors analyzed the proteomes of HeLa cell populations metabolically marked with different stable isotopic forms of arginine, and each population was stimulated by the epidermal growth factor for a various length of time.…”

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

“…Furthermore, methods for tagging with stable isotopes, such as stable isotope labeling in cell culture (SILAC), ICAT, or iTRAQ, provide quantitative information about proteins or peptides from MS or MS/MS spectra [13][14][15][16]. Indeed, the application of these quantitative methods to studies of proteins phosphorylated on tyrosine has identified many new substrates of tyrosine kinases [11,12,17].…”

Stable isotopic labeling in proteomics

“…Heavy labeled variants of lysine and arginine that provide ample spacing between isotopic envelopes of light and heavy tryptic peptides (e.g., 10 Da using 13 C 6 15 N 4 -Arg and 8 Da using 13 C 6 15 N 2 -Lys) are available such that even mass spectrometers with low resolving power (for instance ITs) can be used for quantitative proteomics. Interestingly, since several forms of lysine and arginine are available, they can be "permutated" and thus allow analysis of three different proteomes (e.g., phosphotyrosine proteomes [12]) or more (up to 5-plexing [13]) at the same time.…”