Glycosylation is one of the most important post-translational modifications of proteins and plays a significant role in physiological progresses. Among monosaccharides, sialic acid has special functions due to its structure and position in glycans, and is related to diseases. There are multiple linkage isomers of sialic acids. It is difficult to distinguish them through LC-MS/MS because of their similar chromatographic behaviors and same mass. The SALSA reaction based on ring-opening ammonolysis has been widely applied in released N-glycans and N-glycopeptides, but we have found that reagents for derivatization would lead to condensation between peptides. Therefore, to address this issue, we introduced a strategy called N-SALSA, which involves blocking the N-terminal of the glycopeptide with acetaldehyde or acetaldehyde-13C2 before conducting the SALSA reaction. The optimized N-SALSA reaction has been well applied in standard glycopeptides and glycoprotein bovine fetuin. After 1:1 mixing of the isotopic diethyl labeled glycopeptides, we identified 106 differentially expressed intact N-glycopeptides from syphilis patient serum compared to healthy individuals, successfully achieving the site-specific distinguish of α2,6- and α2,3-linkage isomers through LC-MS/MS.