EasyAb: A High-Throughput Workflow for Antibody-Based PTM Peptide Enrichment Method Coupled to Mass Spectrometry

Abstract

Reversible post-translational modification (PTMs) is a fundamental mechanism of cellular signal transduction. In vivo and ex vivo studies to profile PTMs have greatly advanced our understanding of the complexities of cellular signaling. However, apart from the most commonly studied PTMs, large-scale analysis is still very challenging, limiting our understanding of various cellular processes. PTM-bearing peptides often enriched by antibodies, followed by unbiased mass spectrometry (MS)-based readout of hundreds or thousands of sites. To extend the reach of this powerful technology to in vivo and ex vivo studies with small protein starting amounts, we here developed EasyAb, a streamlined and high throughput MS workflow for antibody-based PTM profiling. Using epidermal growth factor receptor (EGFR) signaling and acute myeloid leukemia (AML) cell systems, we demonstrate that EasyAb increases sensitivity and enables multiple large-scale systems-level studies. Furthermore, EasyAb resolves in vivo brain G protein-coupled receptor-mediated tyrosine kinase activation and reveals the long elusive hypothalamic neuron-specific leptin signaling architecture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/620939v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@259537org.highwire.dtl.DTLVardef@1675a43org.highwire.dtl.DTLVardef@570b61org.highwire.dtl.DTLVardef@ac45df_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSEasyAb, a sensitive, rapid and high-throughput method to quantify post translationally modified peptides in diverse cell systems. EasyAb reveals differential tyrosine phosphorylation of mRNA splicing proteins in AML patient samples. Activation of KOR by "G-protein-biased" non-aversive agonist 6GNTI elicits Src kinase activity in mice. Elucidation of in vivo hypothalamic Leptin induced LEPRb-Jak2 phosphotyrosine signaling architecture.