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-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to improve the mass spectral characteristics of glycoprotein-released 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 onto a probe containing a carbohydrate sample to minimize interferences from cations, anions, and/or detergents introduced from the sample and/or matrix. On-the-probe sample cleanup is fast (a few minutes) and operates best on picomole quantities of analyte in sample volumes less than 5 microliter containing nanomole quantities or less of impurities. This in situ cleanup dramatically increases the mass spectral signal-to-background, improves mass accuracies, better equalizes the sensitivities for diverse carbohydrate structures, and has the potential to remove contaminants that bypassed previous purification schemes. Direct MALDI mass profiling of digest aliquots containing low picomole amounts of carbohydrate structures either enzymatically released from a glycoprotein or sequentially degraded with multiple glycosidases was performed using only microscale digest conditions with reduced buffer amounts and on-the-probe sample cleanup to minimize the digest impurities. Membrane microdialysis was compared to on-the-probe sample cleanup and found to more completely remove the nano- to micromole amounts of anions (and cations) in HPAE fractions in one step as opposed to multiple on-the-probe steps.
Rouse
J C
JC
Genetics Institute, Inc., One Burtt Road, Andover, Massachusetts, 01810, USA.
Vath
J E
JE
D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D003902:Detergents; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases; D017551:Microdialysis; D009439:Neuraminidase; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002241:Carbohydrates; D003902:Detergents; D006023:Glycoproteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D009439:Neuraminidase