Abstract
Analysis of a large number of samples requires an efficient, rapid and reproducible method. Automation is an ideal approach for high-throughput sample preparation. Multi-plexing sample preparation via a 96-well plate format becomes popular in recent years; however, those methods lack specificity and require several cleanup steps via chromatography purification. To overcome these drawbacks, a chemoenzymatic method has been developed utilizing protein conjugation on solid-phase. Previously, sample preparation was successfully performed in a snap-cap spin-column (SCSC) format. However, sample preparation using SCSC is time-consuming and lacks reproducibility. In this work, we integrated the chemoenzymatic technique in a pipette tip (AutoTip) that was operated by an automated liquid handler. We established a multi-step protocol involving protein immobilization, sialic acid modification, and N-glycan release. We first optimized our automated protocol using bovine fetuin as a standard glycoprotein, and then assessed the reproducibility of the AutoTip using isobaric tags for relative N-linked glycan quantification. We then applied this methodology to profile N-glycans from 58 prostate cancer patient urine samples, revealing increased sialyation on urinary N-glycans derived from prostate cancer patients. Our results indicated AutoTip has applications for high-throughput sample preparation for studying the N-linked glycans.
Highlights
Glycosylation is one of the most abundant post-translational modifications (PTM) of proteins
Over 85% fetuin was immobilized to the solid support after 4 h, and over 88% after 6 h, via reductive amination
The results of our study have demonstrated that analysis of a large number of samples is feasible using AutoTip format in conjunction with an automated liquid handler
Summary
Glycosylation is one of the most abundant post-translational modifications (PTM) of proteins. A solid-phase capture method, named glycoprotein immobilization for glycan extraction (GIG), has shown potential for applications in a HTP sample preparation for the analysis of N-linked glycans[25,26,27]. Chemoenzymatic-based methods can be integrated in automated sample preparation platforms for rapid and robust analysis of protein glycosylation. Using an automated liquid handler, up to 96 samples can be processed simultaneously, involving glycoprotein conjugation onto the resin, washing, and subsequent enzymatic digestion: first using trypsin to release non-glycosylated peptides, followed by PNGase F digestion to release the glycosite-containing N-glycopeptides. We designed a novel platform that takes advantage of a pipette tip format (AutoTip), in which aldehyde-functionalized resin is packed, and sample processing is performed by an automated liquid handler. The AutoTip methodology was applied to N-linked glycan analysis of prostate cancer patient urine sample, demonstrating its applicability to N-glycan analysis of glycoproteins from complex biological samples
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