Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes.1 Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation and Ser-phosphorylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically phosphorylated, acetylated, and fluorescently tagged analogs, for the first time.2, 3 The resulting synthetic analogs were employed to decipher the molecular role of Ser-phosphorylation and Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31, and Lys-57 and the phosphorylation at Ser-11 significantly inhibit the DNA binding activity of Max. Furthermore, we found that the acetylation mark can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.4