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The structure of N-glycans on cell surface was abnormal in the process of cancer, which was closely related to the invasion and metastasis of tumors. Total glycopeptides were obtained from trace HeLa cells(10(7)) after cell lyses, reduction and acetamidation, trypsin digestion and Oasis-HLB cartridge purification. N-glycans were released by PNGase F digestion and purified by a Sep-Pak C-18 cartridge. The structural of N-glycans profiling was first analyzed by positive-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) after permethylation. The 34 types of N-glycans obtained from HeLa cell included both high-mannose and complex(bi-, tri-, tetra- and penta-antennary) types, some of the latter containing bisecting and Lewis determinants, which were relevant to tumor metastasis in same way. The MALDI-TOF MS technology was suitable for rapid analysis of N-glycans profiling on cancer cell surface in trace amount, in order to find new glycans-makers and more foundation for prevention and diagnosis in early stage of tumors.