ON-THE-PROBE SAMPLE CLEANUP STRATEGIES FOR GLYCOPROTEIN-RELEASED CARBOHYDRATES PRIOR TO MATRIX-ASSISTED LASER DESORPTION-IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY

On-the-probe sample cleanup strategies were developed for matrix-assis ted laser desorption-ionization (MALDI) time-of-flight mass spectromet ry to improve the mass spectral characteristics of glycoprotein-releas ed carbohydrate samples, including those fractionated by high pH anion exchange (HPAE) chromatography or treated with glycosidases. Small in situ amounts of chromatographic media are codeposited with matrix ont o a probe containing a carbohydrate sample to minimize interferences f rom cations, anions, and/or detergents introduced from the sample and/ or matrix. On-the-probe sample cleanup is fast (a few minutes) and ope rates best on picomole quantities of analyte in sample volumes less th an 5 mu l containing nanomole quantities or less of impurities. This i n situ cleanup dramatically increases the mass spectral signal-to-back ground, improves mass accuracies, better equalizes the sensitivities f or diverse carbohydrate structures, and has the potential to remove co ntaminants that bypassed previous purification schemes, Direct MALDI m ass profiling of digest aliquots containing low picomole amounts of ca rbohydrate structures either enzymatically released from a glycoprotei n or sequentially degraded with multiple glycosidases was performed us ing only microscale digest conditions with reduced buffer amounts and on-the-probe sample cleanup to minimize the digest impurities. Membran e microdialysis was compared to on-the-probe sample cleanup and found to more completely remove the nano- to micromole amounts of anions (an d cations) in HPAE fractions in one step as opposed to multiple on-the -probe steps. (C) 1996 Academic Press, Inc.