Introduction:
Glycoproteins are actively involved in physiological functions as well as certain diseases. However, glycoprotein characterization using native and native top-down mass spectrometry remains challenging as its heterogeneity leading to complex spectrum. In this study, we evaluated a few novel techniques including Direct Mass Technology mode and proton transfer charge reduction (PTCR) to unravel proteoforms of Human Fetuin A, a heavily glycosylated and phosphorylated protein, comprehensively under native condition. We further expand the application to heavily glycosylated Spike protein variants for determining their MW and oligomeric states.
Methods:
Human Fetuin A from human plasma was purchased from Sigma-Aldrich. Fetuin was buffer exchanged into ammonium acetate with Amicon 10K-MWCO or 30K-MWCO. Native MS and Direct Mass Technology mode were performed on Thermo Scientific™ Q Exactive™ UHMR. PTCR and EThcD analyses were performed on Thermo Scientific™ Orbitrap Eclipse™. Data were analyzed using Thermo Scientific™ BioPharma Finder™ 4.1 and STORIboard (Proteinaceous).
Results:
Initial native MS scan of Fetuin shows congested charge envelopes across m/z 3000- 4000. PTCR separated peaks which were previously overlapped in the full scan and thus increased number of identified proteoforms. Isotopically resolved proteoforms unambiguously discloses glycans and phosphorylations combinations. Native top-down analysis of Fetuin using EThcD proves the absence of signal peptide at N-terminus and demonstrates the B-chain is disulfide bonded to the N-terminal of A-chain through cys32-cys358 to form a loop. Top-down analyses of DTT released B-chain could yield 95% sequence and identify partially O-glycosylated site at Ser346. Direct Mass Technology mode analyses of Fetuin cleaned by 30K- and 10K-MWCO indicate different MW distributions with much more glycoforms detected compared to ensemble measurement. Such approach could also obtain MW, impurities, and trimer/monomer states of spike protein variants.
Conclusions:
Complementary mass spectrometry analyses including Native, PTCR, Direct Mass Technology mode, and EThcD could comprehensively characterize heavily glycosylated proteins.