Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification

Janie L. Desantos-Garcia, Sarah I. Khalil, Ahmed Hussein, Yunli Hu, Yehia Mechref

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Aberrant glycosylation of proteins and lipids has been implicated in many human diseases, thus prompting the need for reliable analytical methods that permit dependable quantification of glycans originating from biological specimens. MS of permethylated glycans is currently employed to monitor disease-related aberrant glycosylation of proteins and lipids. However, enhancing the sensitivity of this type of analysis is still needed. Here, analysis of permethylated glycans at enhanced sensitivity is attained through miniaturized solid-phase permethylation and online solid-phase purification. Solid-phase permethylation method was miniaturized by reducing the amount of sodium hydroxide beads (one-third the original amount) packed in microspin columns. The efficiency of glycan permethylation was not adversely affected by this reduction. Online solid-phase purification of permethylated N-glycans derived from model glycoproteins, such as fetuin, α-1 acid glycoprotein and ribonuclease B, offered more sensitive and reproducible results than offline liquid-liquid and solid-phase extractions. Online solid-phase purification method described here permitted a 75% increase in signal intensities of permethylated glycans relative to offline purification methods. This is mainly due to the minimized sample handling associated with an online cleaning procedure. The efficiency and utility of online solid-phase purification was also demonstrated here for N-glycans derived from human blood serum. Online solid-phase purification permitted the detection of 73 N-glycan structures, while only 63 glycan structures were detected in the case of samples purified through liquid-liquid extraction. The intensities of the 63 structures that were detected in both cases were 75% higher for samples that were purified through the online method.

Original languageEnglish
Pages (from-to)3516-3525
Number of pages10
JournalELECTROPHORESIS
Volume32
Issue number24
DOIs
StatePublished - Dec 2011

Keywords

  • Glycans
  • Glycoproteins
  • LC-MS
  • LC-MS/MS
  • Permethylation

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