analyst is faced with a mixture of carbohydrate struc-On-the-probe sample cleanup strategies were develtures to characterize given most glycosylation sites exoped for matrix-assisted laser desorption-ionization hibit structural microheterogenetity (1). Direct analy-(MALDI) time-of-flight mass spectrometry to improve sis of this carbohydrate mixture by matrix-assisted the mass spectral characteristics of glycoprotein-relaser desorption-ionization (2) time-of-flight mass leased carbohydrate samples, including those fracspectrometry (3) (MALDI-TOF-MS) 2 provides molecutionated by high pH anion exchange (HPAE) chromalar masses for many of the structural variants. This tography or treated with glycosidases. Small in situ mass information allows the monosaccharide composiamounts of chromatographic media are codeposited tions of many structures to be identified and indicates with matrix onto a probe containing a carbohydrate whether certain structures have unusual modificasample to minimize interferences from cations, anions, tions.
and/or detergents introduced from the sample and/or
The production of ionized biomolecules by MALDI matrix. On-the-probe sample cleanup is fast (a few requires the incorporation of analyte biomolecules into minutes) and operates best on picomole quantities of the matrix lattice structure during the sample preparaanalyte in sample volumes less than 5 ml containing tion steps (4). Successful analysis of purified biomolenanomole quantities or less of impurities. This in situ cules by MALDI relies on an empirical sample preparacleanup dramatically increases the mass spectral sigtion process that involves optimizing matrix selection, nal-to-background, improves mass accuracies, better matrix-to-analyte ratio, cosolvents, additives, etc. (5).
equalizes the sensitivities for diverse carbohydrate
Impure biological samples present a greater challenge structures, and has the potential to remove contamito MALDI because the incorporation of analyte molenants that bypassed previous purification schemes. Dicules into the matrix crystals competes with a sea of rect MALDI mass profiling of digest aliquots containing low picomole amounts of carbohydrate buffer salts, detergents, denaturants, metal chelating structures either enzymatically released from a glyco-agents, ion-paring agents, etc. A recognized benefit of protein or sequentially degraded with multiple glycos-MALDI is the tolerance of low levels of these impuriidases was performed using only microscale digest ties, but higher levels ultimately require an additional conditions with reduced buffer amounts and on-thecleanup step. The motivation for removing these impuprobe sample cleanup to minimize the digest impuririties from a heavily or even a mildly impure sample ties. Membrane microdialysis was compared to on-theis to improve the mass spectral characteristics such as probe sample cleanup and found to more completely signal-to-background, sensitivity, resolution, and mass remove the nano-to micromole amounts of anions (and accuracy, and to allow for direct MALDI sample analycations) in HPAE fractions in one step as opposed to sis without time-consuming sample cleanup.
multiple on-the-probe steps.