THE USE OF MULTIDIMENSIONAL LIQUID-PHASE SEPARATIONS AND MASS-SPECTROMETRY FOR THE DETAILED CHARACTERIZATION OF POSTTRANSLATIONAL MODIFICATIONS IN GLYCOPROTEINS

The goal of characterization of the proteome, while challenging in its elf, is further complicated by the microheterogeneity introduced by po sttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass s pectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificit y and sensitivity of MS. In the current work, the combination of liqui d-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionizatio n time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields re al-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide fami ly was used to develop an integrated multidimensional analysis achieve d by the off-line coupling of LC, CE, and MALDI TOF-MS, To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoprote ins, we will report on the study of the glycoforms present in a N-link ed site in a single-chain plasminogen activator (DSPA alpha 1).