Ultraviolet photodissociation (UVPD) is a powerful tool for top-down proteomics due to the high efficiency and indiscriminant nature of its fragmentation. While UVPD has been demonstrated for glycopeptide and glycan analysis, it has not yet been tested on intact glycoproteins. Here we demonstrate the utility of top-down UVPD for analyzing both the composition and locations of glycosylations
Disulfide intact and reduced and alkylated glycoprotein ions were produced by static nanoelectrospray ionization from denaturing solutions of 49/50/1 water/methanol/acetic acid. Ions were analyzed on a Thermo Orbitrap Fusion Lumos Tribrid MS, and top-down fragmentation was performed with HCD, ETD, or ultraviolet photodissociation at 213 nm for each ion.
UVPD fragmentation of disulfide reduced proteins produced predominantly a and x ions and preferential fragmentation at proline residues, consistent with previous observations for glycopeptides. UVPD of disulfide reduced ribonuclease B resulted in single amino acid resolution for the site of the glycan, and fragment ions were composed predominantly of cleavage along the protein backbone with retention of the entire glycan, whereas UVPD of disulfide intact ribonuclease B resulted in predominantly cleavage of the entire glycan from the precursor and charge reduced precursor ions, and close to 100% sequence coverage for the termini of the proteins up until the locations of the disulfide bonds. This data demonstrates that complementary information can be gained from top-down UVPD of folded and unfolded glycoproteins -- the exact masses of the glycans from UVPD of the folded form of the protein, and the residues on which those glycans are located from UVPD of the unfolded form of the protein.
This is the first demonstration of the power of UVPD for top-down analysis of glycoprotein