Glycoproteins are gaining attention as they are actively involved in physiological functions and in the progression of certain diseases. However, glycoprotein characterization using intact and top-down mass spectrometry remains challenging as its heterogeneity leads to complex spectra. This study leverages proton transfer charge reduction (PTCR) and Direct Mass Technology mode (charge detection mass spectrometry, CDMS) on Orbitrap platforms to unravel glycoprotein complexity at the intact level.
Glycoproteins were buffer exchanged into ammonium acetate with Amicon 10K-MWCO or 30K-MWCO. Native MS and Direct Mass Technology were performed by direct infusion with a Nanoflex ion source coupled to Thermo Scientific™ Q Exactive™ UHMR MS. Data independent acquisition-Proton Transfer Charge Reduction (DIA-PTCR) was performed on Orbitrap Ascend modified with a high quadrupole mass filter up to m/z 8000. The complex glycoproteins were quadrupole-isolated in narrow windows to minimize interference from various glycoform signals. Subsequently, the isolated ion packets were charge reduced in the ion trap and scanned in the Orbitrap. EThcD fragmentation was used to confirm sequence, determine glycosylation, and resolve chain linkage. Data were analyzed using Thermo Scientific™ BioPharma Finder™ 5.2 Software and STORIboard processing software (Proteinaceous).
Both recombinant human Fetuin and spike protein exhibit a plethora of PTMs. To confirm sample identity and assess the impurity present, we first performed native top-down analysis of glycoproteins using EThcD. To benchmark the newly developed DIA-PTCR method we performed PTRC analysis using 5-20Th isolation windows. Preliminary data demonstrates that 5 Th isolation of Fetuin followed by PTCR could reduce the charge state from +12 to +4. Moreover, we successfully separated two distinct charge envelopes originating from precursors, each representing unique PTMs. Stepping through m/z 3300-4500 with 10 Th isolation prior to PTCR unveiled numerous glycoforms from 38 kDa to 46 kDa. The ultra-high resolution at 480K enables the isotope resolution of each glycoform, resulting in unambiguous monoisotopic MW. A full MS scan of spike protein displayed partially resolved peaks atop the elevated baseline. Employing the DIA-PTCR across the entire m/z range could not only resolve the charge states of glycoforms decorated with various PTMs e.g., glycosylation and phosphorylation, but also identify their dimers near 100 kDa. Surprisingly, we also observed a cluster of peaks ~70 kDa. These findings are consistent with complementary Direct Mass technology (CDMS) analyses.
By combining different MS methods such as Direct Mass Technology, DIA-PTCR and EThcD, we could successfully characterize heavily glycosylated proteins while also revealing sample contaminants.