High-throughput solid-phase permethylation of glycans prior to mass spectrometry.
Permethylation of glycans prior to their mass spectrometric determination has now become a time-honored methodology in glycoconjugate analysis due to the advantage of a simultaneous analysis of neutral and acidic glycans as well as enhanced sensitivity and easier tandem mass spectrometry interpretation. While the different solvent extraction-based versions of this method often suffice in different structural studies, they are generally less satisfactory in the quantitative determinations aiming at minor quantities of the analyzed materials. To overcome these difficulties, we recently introduced a solid-phase capillary permethylation technique (Kang et al., Rapid Commun. Mass Spectrom. 2005; 19: 3421) for microscale determination. Here, we describe a very useful high-throughput extension of the solid-phase methodology utilizing spin columns packed with sodium hydroxide beads. This procedure has been thoroughly optimized to match the analytical performance parameters of the previously used capillary technique. As demonstrated with a high-precision glycomic profiling analysis of human blood serum, this methodological improvement offers simplicity and reproducibility, allowing the complete permethylation of 12-18 samples in less than 20 min.
Kang
Pilsoo
P
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Mechref
Yehia
Y
Novotny
Milos V
MV
D006801:Humans; D008745:Methylation; D008832:Microchemistry / Q000379:methods; D011134:Polysaccharides / Q000737:chemistry*; D040901:Proteomics; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011134:Polysaccharides