Chen et al synthesized a long fragment of Shc1 with different phosphorylation motifs and applied the synthetic products in affinity-purification mass spectrometry (AP-MS) to identify phosphorylation-dependent interacting proteins of Shc1. By comparing the difference in spectral counting after AP-MS, they identified several potential targets that are specifically binding the phosphorylated forms of Shc1. They also demonstrated that the synthetic proteins could be used a diagnostic probe for differentiating RTK-signaling networks in different breast cancer cells. The study showcased the power of protein synthesis in creating PTM-dependent proteoforms of target of interest and the strength of AP-MS to identify complexed signaling networks in cellular proteomes. However, the data quality and presentation need further improvement before it is considered for publication. Here are a couple of major comments:

1) Most of the main figures are presented with volcano plots and the study lacked sufficient biochemical validation. For example, a major target should be validated by western blotting to show it can be pulled down with difference by the phosphorylation-dependent fragments. More convincingly, a reciprocal pulldown should be performed to show that a specific interacting protein, such as GRB2, can pull down the Shc1 fragments with different affinities. Without such cross validation, it is hard to evaluate the overall all quality of the proteomic data.
2) A molecular docking study is suggested to explain why the different phosphorylation proteoforms can bind their interacting protein differently. With AlphaFold3 being published, it can be applied to investigate structurally how phosphorylation can affects the specific interactions between Shc1 and its interactor proteins.
3) The figures are generally made with low quality and the overall writing also need improvement with logical clarity.

The authors have done a good job in revising and improving the manuscript. I support its publication