Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Abstract

The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-a were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity (,25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetrasialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.

Effect of sialic acid content on glycoprotein pI analyzed by two‐dimensional electrophoresis

“…Many authors have studied the glycosylation features of human and recombinant EPOs in order to distinguish them due to both the misuse of rEPOs among athletes and to the clinical use of these drugs as a treatment for anaemia [16,18,22,23]. Several spots corresponding to rEPOs are resolvable at pIs between 3 and 5, depending on the source [22][23][24][25].…”

“…Many authors have studied the glycosylation features of human and recombinant EPOs in order to distinguish them due to both the misuse of rEPOs among athletes and to the clinical use of these drugs as a treatment for anaemia [16,18,22,23]. Several spots corresponding to rEPOs are resolvable at pIs between 3 and 5, depending on the source [22][23][24][25]. Different studies have concluded that the trains of spots observed in 2-DE for EPO (both human and recombinant) are a result of different sialylation patterns of EPO, but none of these studies have obtained a correlation between the pI and the SA content [16,18,22,23].…”

“…Several spots corresponding to rEPOs are resolvable at pIs between 3 and 5, depending on the source [22][23][24][25]. Different studies have concluded that the trains of spots observed in 2-DE for EPO (both human and recombinant) are a result of different sialylation patterns of EPO, but none of these studies have obtained a correlation between the pI and the SA content [16,18,22,23]. According to one study [18], the total average content of SA of rEPO (Neorecormon s ) is 13 mol/mol EPO, and the pI shift after sialidase treatment is approximately 3.1-4.1 pH units.…”

“…Thus, the contribution of SA to this pI shift can be calculated as 3.1/13 pI units per sialic acid molecule (pI/SA) -4.1/13 pI/SA 5 0.24-0.32 pI decrease per SA molecule. Full characterisation of the SA content of each rEPO spot resolved by 2-DE has already been performed [23], but the pI was not assigned to each spot making it impossible to calculate the correlation between the SA content and the pI.…”

MS Platform for Erythropoietin Glycome Characterization

“…Rapid glycan profiling is possible with few seconds of acquisition times in a high-throughput manner. 4,5,20 Most of EPO N-glycans are multi-antennary glycans having high sialic acid residues, therefore, EPO Nglycans are analyzed in the negative ion detection mode to obtain overall profiling of N-glycan compositions during MALDI-MS. Figure 2a shows representative MALDI-TOF MS spectrum of native N-glycans released from the second generation EPO (NESP). High resolution TOF-MS provided accurate mass, simplifying identification of molecular ion, compositional assignment of each detected glycans.…”