Tyrosine Phosphoproteomics of Fibroblast Growth Factor Signaling

Abstract

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor. Fibroblast growth factors (FGFs)1 stimulate differentiation, proliferation, and other cellular events by inducing activation of the intrinsic tyrosine kinase in membrane-spanning receptors of the FGF receptor (FGFR) family. As receptors become activated, they autophosphorylate on several residues outside the kinase domain, thus allowing recruitment of cytosolic substrates and initiation of multiple downstream signaling pathways in parallel. The FGFR has been shown to initiate signaling through direct binding of substrates such as phospholipase C␥ (PLC␥) (1, 2), FGFR substrate-2 (FRS2) (3, 4), and ShcA (5). The end point of FGFR activation is defined in a cell-specific manner by the integration of these signaling pathways. A thorough understanding of cellular signaling processes requires the identification of the components of the entire signaling network followed by studies of the functional role of individual proteins. Large scale analysis of post-translationally modified (e.g. phosphorylated) proteins by mass spectrometry (MS) has gained momentum with the development of high resolution and high sensitivity hybrid mass spectrometers (e.g. quadrupole time-offlight (qTOF) instruments) and now constitutes a powerful functional proteomics platform for the analysis of protein networks of signaling processes (6).Since signaling proteins are not all "turned off" in the quiescent cell, and the stoichiometry of activation may vary according to the stimulu...

Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway

“…Relative quantification ratios of the identified phosphopeptides were derived by MSQuant as previously described (38). Briefly peptide ratios between the monoisotopic peaks of "normal" and "heavy" forms of the phosphopeptide were calculated and averaged over consecutive MS cycles for the duration of their respective LC-MS peaks in the total ion chromatogram using both FT survey and FT SIM scans.…”

Proteomic Mapping of Brain Plasma Membrane Proteins

“…To make our method applicable to identification of membrane proteins using minute amounts of biological material, we miniaturized the sample preparation protocol and applied a recently developed hybrid linear ion trap-FT mass spectrometer (LTQ-FT) (19). This instrument is significantly more sensitive, accurate, and has higher sequencing speed compared with a Q-TOF (20). Hippocampus from a single mouse (10 -20 mg) was dissected from frozen brain.…”

Protein Phosphorylation and Expression Profiling by Yin-Yang Multidimensional Liquid Chromatography (Yin-Yang MDLC) Mass Spectrometry

“…Some high-affinity chemical derivation methods [15][16][17][18] have emerged, but suffer from side reactions, marginal yields, and low specificities. Immunopurification [19][20][21][22] and affinity chromatography including immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) are frequently used techniques for the enrichment of phosphorylated proteins and peptides, [23][24][25][26][27][28] but also suffer from unsatisfactory specificity or sensitivity. Recently, strong-cation exchange (SCX) chromatography using salt gradient has been applied for phosphopeptide enrichment by collecting fractions from an SCX column followed by reverse-phase chromatography and mass spectrometry.…”