ABSTRACT: Abstract
Glycopeptide Abundance Distribution Spectra (GADS) were recently introduced as a means of representing, storing and comparing glycan profiles of intact glycopeptides. Here, using that representation, an extensive analysis is made of multiple commercial sources of recombinant SARS-CoV-2 spike protein, each containing 22 N-linked glycan sites (sequons). Multiple proteases are used along with variable energy fragmentation, followed by ion trap confirmation. This enables a detailed examination of reproducibility of the method across multiple types of variability. These results show that GADS are consistent between replicates and laboratories for sufficiently abundant glycopeptides. Then derived GADS enable the examination and comparison of the glycan profiles between commercial sources of the spike protein. Throughout multiple distinct glycopeptide distributions, generated by multiple proteases, confirm these profiles. Comparisons of GADS derived from 11 sources of recombinant spike protein reveal that sources for which protein expression methods were the same produced near-identical glycan profiles, thereby demonstrating the ability of this method to measure GADS of sufficient reliability to distinguish different glycoform distributions between commercial vendors and potentially to reliably determine and compare differences in glycosylation for any glycoprotein under different conditions of production. GADS libraries for all 11 sources of recombinant spike protein have been made available for download (https://chemdata.nist.gov/dokuwiki/doku.php?id=peptidew:sars-cov-2_spike_protein_n_linked_glycoLC-MS/MS Analysis ).
LC-MS/MS Analysis
Spike protein digests were analyzed on an UltiMate 3000 (Thermo Fisher Scientific) in-line with an Orbitrap Fusion Lumos (Thermo Fisher Scientific) mass spectrometer. Reversed-phase separation was performed on an Acclaim PepMap RSLC 75 um x 75 cm column (Thermo Fisher Scientific) at a flow rate of 200 nL/min. The gradient consisted of (min: % Solvent B): 0:2, 320:32, 366:80, 383:98, 396:98, 409:2, 490:2, where solvent B is 0.1% (v/v) formic acid in acetonitrile.
Data-dependent acquisition was performed in positive ion mode with an ion transfer tube temperature of 275 C and a spray voltage of 1.80 kV. MS survey scans, or MS1 scans, were acquired with a scan range of m/z 380-2000, a maximum injection time of 50 ms, AGC target of 400 000 and RF Lens value of 40%. Precursor ions were detected in the orbitrap with a resolution of 120 000. Precursor ions selected for fragmentation included those with charge states 2-8 with a minimum intensity of 50 000. A dynamic exclusion of 15 seconds was used with a tolerance of +/- 10 ppm. Tandem mass spectra were acquired with a cycle time of 5 seconds in between MS1 scans, a maximum injection time of 60 ms, an AGC target of 50 000 and a fixed starting m/z of 120. Beam-type collision cell spectra (HCD) using stepped collision energies of NCE 15, 25 and 35% were acquired and detected in the orbitrap with a resolution of 30 000. An ion trap scan was triggered if m/z 204.087 (15 ppm tolerance) was among the top 20 product ions in the HCD scan. The ion trap scan was acquired with a collision energy of 30%, activation Q of 0.25 and activation time of 10 ms. Product ions were detected in the orbitrap with a resolution of 30 000.
Data Analysis
Glycopeptide assignments were made using Byonic and Byologic software (version 3.10 Protein Metrics, Inc.). For each protease, with the exception of alpha lytic proteases, up to three missed cleavages were allowed using cleavage rules listed in Table 2. Up to six missed cleavages were allowed for alpha lytic wild type and mutant proteases. Assignments were made using precursor and product ion tolerances of 5 and 20 ppm, respectively. Cysteines were required to contain a fixed carbamidomethyl while variable modifications included oxidation of methionine and N-terminal pyroglutamine. For glycopeptides, a library containing 445 N-glycans was used (Supplemental Table S1).
Meghan Burke 
