The combination of distinct peptide ligation techniques to facilitate chemical protein synthesis represents one of the long-standing goals in the field. A new combination ligation method of N-to-C sequential native chemical ligation and Ser/Thr ligation (NCL-STL) is described for the first time. This method relies on the peptide salicylaldehyde S,S-propanedithioacetal (SALPDT)-ester prepared by a new 1,3-propanedithiol-mediated reaction. The peptide SALPDT-ester, which is compatible with NCL, can be fully activated by N-chlorosuccinimide (NCS)/AgNO3 in aqueous solution to afford peptide SAL-ester for use in the subsequent STL. The practicality of the combined NCL-STL method is illustrated by the synthesis of S-palmitoylated matrix-2 (S-palm M2) ion channel from Influenza A virus and S-palmitoylated interferon-induced transmembrane protein 3 (S-palm IFITM3). This approach expands the multiple-segments peptide ligation toolkit for producing important and complex custom-made protein samples by chemical protein synthesis.