Qualitative and Quantitative Analysis of Glycans Released from Model Glycoproteins and Pooled Human Blood Serum using MRM Mode and ESI LC-MS/MS



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Protein glycosylation is one of the most common posttranslational modifications (PTM). Aberrant glycosylation has been correlated with various mammalian diseases. Thus, qualitative and quantitative strategies are necessary to monitor glycan changes correlated with different biological conditions. One of the most effective quantitative strategies for reliable and sensitive monitoring of glycan changes is mass spectrometry (MS). However, development of reliable sample sample preparation methods and optimization of analysis conditions is required to monitor the changes associated with disease states. The 2nd chapter describes an enhanced sensitivity through miniaturized solid-phase permethylation and online solid-phase permethylation. 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, a-1 acid glycoprotein and ribonuclease B, offered more sensitive and reproducible results than offline liquid–liquid and solid-phase extractions. The 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. vii The 3rd chapter introduces the quantification of N-linked glycans using multiple reaction monitoring (MRM) on a triple quadrupole instrument. The optimized collision energy (CE) for sialylated and fucosylated N-glycans was 35 ev and the CE for mannose, and complex type N-glycans was 30 ev. In addition to the optimized CE, the selection of 3 transitions was also established for reliable quantification. A total of 88 N-glycans in human blood serum were quantified using a multiplexed MRM approach with these optimized conditions. Reliable detection of these N-glycans measured at 0.005 μL indicates that N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, yielding a dynamic concentration range of 3 orders of magnitude in a single experiment. Also, MRM was used to identify the N-glycans differentially expressed in brain-targeting breast carcinoma cells (MDA-MB-231-Br) and was compared with MDA-MB-231 breast cancer cell line. Thus, MRM measurements provide a rapid and reliable identification and quantification of glycans that could be applied in biomarker discovery studies.