LC-MS/MS-PRM Quantification of IgG Glycoforms Using Stable Isotope Labeled IgG1 Fc Glycopeptide Standard

Targeted quantification of proteins
is a standard methodology with
broad utility, but targeted quantification of glycoproteins has not
reached its full potential. The lack of optimized workflows and isotopically
labeled standards limits the acceptance of glycoproteomics quantification.
In this work, we introduce an efficient and streamlined chemoenzymatic
synthesis of a library of isotopically labeled glycopeptides of IgG1
which we use for quantification in an energy optimized LC-MS/MS-PRM
workflow. Incorporation of the stable isotope labeled N-acetylglucosamine
enables an efficient monitoring of all major fragment ions of the
glycopeptides generated under the soft higher-energy C-trap dissociation
(HCD) conditions, which reduces the coefficients of variability (CVs)
of the quantification to 0.7–2.8%. Our results document, for
the first time, that the workflow using a combination of stable isotope
labeled standards with intrascan normalization enables quantification
of the glycopeptides by an electron transfer dissociation (ETD) workflow,
as well as the HCD workflow, with the highest sensitivity compared
to traditional workflows. This was exemplified by a rapid quantification
(13 min) of IgG1 Fc glycoforms from COVID-19 patients.