As a critical adaptor protein, Shc1 contains SH2 and PTB domains for scaffolding tyrosine phosphorylation-dependent protein complexes, therefore regulating growth factor receptor signaling networks. The three tyrosine phosphorylation sites (pY) on the CH1 linker region of SHC1 provide additional docking sites for recruiting additional dimension of signaling complexes. The affinity purification-mass spectrometry (AP-MS) method with synthetic phosphopeptides provides an unbiased discovery approach but is often limited by the length of synthetic peptides. Here, we chemically synthesized the CH1 region (Shc1CH1) covering 107 amino acids with the 239, 240, and 313 pY sites by multiple chemical peptide ligations. Combing with AP-MS and accurate label-free quantification, we explored the site-specific interactome of these distinct phospho-forms of Shc1 and identified the enhanced complex assembly around the double-phosphorylated Y239/204 sites. We also found that Plcg1 and Plcg2 interacted with pY313 strongly and specifically. Besides, we demonstrated that the AP-MS with the mouse Shc1CH1 probes could evaluate the subcellular EGFR/HER2 signaling in three human cell lines. Overall, long synthetic peptides assembled via peptide ligation widened the scope of peptide-based AP-MS approach for the analysis of site-specific interactomes of proteins with long phosphorylated docking sites.