Aberrant glycosylation of proteins and lipids has been implicated in many human diseases, thus prompting the need for reliable analytical methods that permit reliable 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 as well as on-line 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. On-line 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 off-line liquid-liquid and solid-phase extractions. On-line solid-phase purification method described here permitted a seventy-five percent increase in signal intensities of permethylated glycans relative to off-line purification methods. This is mainly due to the minimized sample handling associated with an on-line cleaning procedure. The efficiency and utility of on-line solid-phase purification was also demonstrated for N-glycans derived from human blood serum. On-line solid-phase purification permitted the detection of 66 N-glycan structures, while only 58 glycan structures were detected in the case of samples purified through liquid-liquid extraction. The intensities of the 58 structures that were detected in both cases were seventy-five percent higher for samples that were purified through the described method.

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and require special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to on-line purification and LC-ESI-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-µl aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak area of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs. mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.

A 75-year-old male patient with a history of previous right lung transplant presented with left upper lobe squamous cell carcinoma. Endobronchial ultrasound and positron emission tomography displayed no mediastinal lymphadenopathy. A ventilation-perfusion scan displayed minimal perfusion to the native lung. Left robot-assisted lysis of adhesions, decortication, left upper lobectomy, and mediastinal lymphadenectomy were performed. The patient tolerated the procedure well. Final pathology displayed pT2a, n0, m0. Lobectomy is a safe and efficient treatment of native lung malignancy in the setting of previous lung transplant with minimally functioning native lung.

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