Effect of 1-phenyl-3-methyl-5-pyrazolone labeling on the fragmentation behavior of asialo and sialylated N-linked glycans under electrospray ionization conditions

The advantages of labeling free N-linked oligosaccharides with 1-phenyl-3-m ethyl-5-pyrazolone (PMP), for high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) are discussed. The study focuses on some asialo and sialylated sugars, and compares the HPLC a nd ESI-MS behaviors of the PMP-labeled substances vs. the native compounds. It is pointed out that native free N-linked carbohydrates have very low af finities for the Cls reversed phases commonly used in HPLC. Native asialo o ligosaccharides yield good ESI-MS sensitivity, although they are very susce ptible to in-source collision-induced dissociation (CID), and the fragments are produced from any of the branches of the molecules, i,e, do not give s pecific structural information. Native N-linked standards bearing one siali c acid residue yield a III-fold loss of ESI-MS sensitivity vs. asialo compo unds, and native sugars with two sialic acid moieties were not detectable. The PMP labeling of asialo and sialylated sugars yielded higher affinities for HPLC Cls columns and, even at the early stages of method development? i t was possible to separate three PMP-labeled standards to a useful extent. In ESI-RIS, PMP-asialo sugars did not yield a significant increase in sensi tivity vs. the native species; however, fragmentation produced by in-source CID was more directed as all predominant fragment ions contained the bis-P MP label. This feature is particularly useful when structural determination of an unknown sugar is required. PMP-sialylated sugars gave rise to very c lean and informative ESI mass spectra, The monosialo sugar yielded a 100-fo ld sensitivity improvement vs. its native analog and, in the case of the di sialylated compound, a 100% improvement was obtained in the positive made. Most fragment ions were informative and contained the reducing end on the m olecules, thus facilitating spectral interpretation. The combination of PMP derivatization with on-line HPLC/ESI-MS is a promising method for the anal ysis of asialo and sialylated carbohydrate mixtures. Copyright (C) 1999 Joh n Wiley & Sons, Ltd.