Swift Universal Glycan Acquisition (SUGA) enables quantitative glycan composition profiling across diverse sample types
Creators: name: Ashwood, Christopher
name: Voelker, Cecilia
name: Cummings, Richard D
Publisher: name: Zenodo
Year: 2022
Subjects: glycomics, mass spectrometry, glycans, glycobiology

description: <strong>Introduction</strong> Protein glycosylation is the most frequent and varied type of co- and post-translational modification seen in eukaryotic cells. Identification of the glycan compositions that modify individual proteins (<em>e.g.</em> therapeutic proteins) or protein mixtures (<em>e.g.</em> cell lysates and plasma) is valuable for hypothesis generation regarding the roles these glycans play in cellular physiology. As sample preparation to obtain released glycans approaches 96- and 384- well plate formats, advances in the throughput of data acquisition have been lacking. Here, we describe a facile flow injection analysis mass spectrometry method that enables profiling of a wide range of glycoconjugates with a total runtime of 3 minutes per sample, while achieving equivalent, or better, data quality compared to MALDI-based data acquisition. <strong>Methods</strong> <em>N</em>-glycans, <em>O</em>-glycans, and a glucose polymer ladder were prepared in 96-well plate formats for SUGA optimization, harmonization of data acquisition to MALDI, and method benchmarking. Glycans were prepared and analyzed in both native and permethylated formats to specifically optimize methods for negative and positive mode, respectively. Pump and autosampler parameters were optimized for repeatability, generating a 1-minute-wide sample plug for MS analysis. Data acquisition methods were developed on a Orbitrap Fusion Lumos Tribrid to achieve the highest possible glycan signal intensity and optimal MS2 quality. These methods enable near-continuous ion stream sampling for a minimum of 10 MS1 scans and 24 MS2 scans per sample plug, surpassing the ideal number of points across a peak for quantitative accuracy. <strong>Preliminary Data</strong> Ten solvents were screened for positive and negative mode ionization efficiency and spectral quality. Piperidine and formic acid were identified as the optimal solvents for native and permethylated glycan acquisition, respectively. Instrument parameters, including resolution, maximum ion injection time, m/z range and, numbers of scans to be averaged, were optimized to obtain the highest signal-to-noise ratios possible for typical glycomic samples. This optimized method enabled glycan compositional profiling over three and a half orders of magnitude for dextran ladder ranging from five to 36 glucose residues, in a total acquisition time of three minutes per sample. This is one of the highest throughputs reported for glycomics, enabling up to 480 samples to be analyzed per day. For the purposes of benchmarking SUGA performance compared to previously published methods for glycan compositional profiling, <em>N</em>-glycans released from glycoprotein standards featuring various glycan classes were acquired by MALDI-TOF and the SUGA method. Glycan intensity correlated overall (0.9 R<sup>2</sup>), however, sulfated <em>N</em>-glycans were detected best with the SUGA method, likely owing to the multiply charged precursor species generated by ESI. These multiply charged precursors also improved MS2 quality, generating more informative fragments for multiple glycan classes which may prove useful for future efforts towards structural elucidation. The quality of spectra generated by native, and permethylated glycans was interrogated by direct comparisons using the same amount of starting glycan material. While permethylated glycans had an overall higher intensity, and subsequently lower maximum ion injection time, the signal quality of the natively analyzed glycans was higher. In-source fragments of native glycans were not detected. Investigations as to the cause of this improved spectral quality are ongoing. The quantitative performance of the method was assessed by defined mixtures of glycan compositions acquired by SUGA. Observed fold changes of glycans matched the expected, demonstrating accuracy and repeatability. <strong>Novel Aspect</strong> The first automated, high throughput ESI method for high quality MS1 and MS2 acquisition of glycan compositions