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

Abstract

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.

Keywords: Glycopeptide Synthesis; Glycoproteomics; Immunoglobulins; Mass Spectrometry; PRM Analysis.

Scheme 1. Synthesis of the ¹³C-Labeled N-Acetylglucosamine (GlcNAc)–Peptide Precursors (A) and Different N-Glycan Oxazolines (B)

Scheme 2. Chemoenzymatic Synthesis of Various Isotope Labeled IgG1 Fc Glycopeptides

Figure 1

Selectivity of glycopeptide fragments recorded under different CE conditions: Low CE condition (NCE 11), signal of antenna loss Y fragment ion (left); high collision energy (NCE 35), signal GlcNAc peptide Y fragment ion; high collision energy (NCE 35) signal of HexNAcHex oxonium ion.

Figure 2

Comparison of the intensities of the most intense soft fragment (low CE) and peptide-HexNAc fragment (high CE) obtained under the following conditions: (A) low NCE, 10 Da window with (C) zoom of qualification ions; (B) high NCE, 10 Da window with (D) zoom of quantification ions.

Figure 3

Comparison of the intensities and RSDs of the quantification of three IgG glycoforms (G0F, G1F, and G2F) in the samples of unfractionated human serum. We used HCD with low (11) and high (35) NCE as well as narrow (1.6 Da) and wide (10 Da) window as described in the legend.

Figure 4

Selectivity of the (A, C) intrascan (10 Da) and (B, D) interscan (1.6 Da) normalization methodology recorded under high and low CE HCD; (C, D) extracted ion chromatography (XIC) signal of peptide-HexNAc Y ion and (A,B) low CE signal of antenna loss Y ion.