Analysis of Glycoproteins by ATR-FTIR Spectroscopy: Comparative Assessment

Probing Blood Plasma Protein Glycosylation with Infrared Spectroscopy

“…After IEX separation ( Figures 2 a and 3 a), we collected each chromatographic peak into a separate fraction and recorded their IR spectra ( Figures 2 b and 3 b). The spectra were normalized to the absorption of the protein backbone (1476–1718 cm –1 ), 18 and spectroscopic global indices of glycosylation were computed as the integral absorption in the carbohydrate region (1000–1180 cm –1 ), following the definition proposed by Derenne et al 18 (see Experimental Section ). We find that the spectroscopic global indices of glycosylation consistently grow with elution time ( Figure 2 c and 3 c), suggesting that heavily glycosylated proteoforms are more acidic than those with a lower global degree of glycosylation and therefore elute later in time.…”

“…14−16 Although the overlap between the absorption bands of different glycan moieties hinders their identification, IR spectra of intact proteins are sensitive to a change in degree of glycosylation and saccharide composition (i.e., micro-and macroheterogeneity). 13,17,18 Previously, infrared spectroscopy was used to evaluate glycosylation of proteins in pioneering work in 2005− 2006, 13,19 but only recently, the understanding of biological importance of glycosylation prompted further research in this direction. 20−22 For instance, IR spectroscopy has been proposed as a tool to control glycosylation of therapeutic monoclonal antibodies.…”

“…Calculation of the Spectroscopic Global Glycosylation Index. Following the algorithm proposed by Derenne et al, 18 we calculated the global glycosylation level based on the spectroscopic data. First, we recorded and processed the FTIR absorption spectra of the protein or the glycoform as described above.…”

“…Therefore, intact proteins can be investigated, eliminating the need for protein digestion or glycan release, and all types of glycans (N-, O-glycans) can be studied simultaneously. Moreover, IR spectroscopic measurements are experimentally straightforward, with short measurement time, performed at low cost, and highly reproducible. − Although the overlap between the absorption bands of different glycan moieties hinders their identification, IR spectra of intact proteins are sensitive to a change in degree of glycosylation and saccharide composition (i.e., micro- and macroheterogeneity). ,, …”

“… 14 − 16 Although the overlap between the absorption bands of different glycan moieties hinders their identification, IR spectra of intact proteins are sensitive to a change in degree of glycosylation and saccharide composition (i.e., micro- and macroheterogeneity). 13 , 17 , 18 …”