<i>N</i> -Glycosylation differences between recombinant and wildtype glycoproteins revealed by mass spectrometry

Mass spectrometry (MS) has emerged as a powerful technique to study protein glycosylation. MS on intact denatured or native proteins can reveal all-inclusive glycoproteoform profiles while top-down, middle-down and/or bottom-up MS can uncover the characteristics of individual glycosylation sites. Alternatively, analyzing enzymatically released <i>N</i>-glycans can reveal intricate details on glycan isomers and generate high-throughput data on larger cohorts. All these methods are increasingly applied for the study of both individual glycoproteins and complex glycoprotein mixtures such as those originating from blood plasma or cell lysates. This has increased our knowledge about the complexity of protein glycosylation, but also revealed its huge diversity, which depends not only on the protein but also on the cell-dependent glycosylation machinery that may change with physiological conditions. Currently, multiple glycoproteins are recombinantly produced, for therapeutic applications as well as in the food sector, in host cells of diverse origin, most commonly: <i>E. coli</i> bacteria, yeast cells, insect cells, mammalian CHO or human HEK293 cells. Although glycoproteins of interest might show similar yields when produced in different host cells, an important question remains whether the host cell will or can provide similar or alike glycoproteoform profiles. In this review, we focus on the application of MS-based technologies to study glycosylation profiles of endogenous human glycoproteins and their recombinantly produced counterparts in different host cells. We will discuss in which ways recombinant glycoproteins can differ from their endogenous variants, and the functional consequences.