## 3 Results and Discussion
### Selective Enrichment of Glycoprotein and Glycopeptides  
In order to prove the high reactivity between alkoxyamine and aldehyde groups, we evaluated the ability of MSP@PNAAMm to enrich the model glycoproteins. In general, for glycoproteins, the diol structure of saccharide can be oxidized to aldehyde groups, which can bond with the alkoxyamine group. RNB, a typical mono-N-glycosylation protein was chosen as the model glycoprotein and the MSP@PNAAMm-1 as the enrichment substrate. The mechanism of enrichment was shown in Scheme 3 and the results were shown in Figure 7. In Lane 1, it was the typical band of protein RNB. After oxidation and conjugation, all of the RNB disappeared in the supernatant (Lane 2). Then with the catalysis of PNGase F, the deglycosylated RNB was eluted in Lane 3. The glycoprotein band with the different molecular weight from Lane 1 represented the lost saccharide structure of RNB by PNGase F. The selectivity of glycoprotein was investigated subsequently by enriching glycoproteins in a mixture with non-glycoproteinins (BSA and LYS). The results were shown in Figure 7, Lane 4 to Lane 6. Before enrichment, all the proteins were shown in Lane 4. After oxidation and conjugation, BSA and LYS still remained in the supernatant, but the RNB disappeared in Lane 5. In Lane 6, only deglycosylated RNB was found. There results prove that the MSP@PNAAMm could be applied to enrich glycoproteins selectively, and this property provides a potential ability to enrich glycoproteins and glycoptides selectively in a complex system. **Scheme 3. ** Mechanism of glycoprotein and glycoptide enrichment with magnetic core-shell microspheres (MSP@PNAAMm-1). Figure 6: TEM images of (**a**) MSP@PNAAMm-_p-co_-PNIPAm; (**b**) MSP@PNAAMm-_p-co_-PAA; (**c**) MSP@PNAAMm-_p-co_-HEMA. The scale bar is 100 nm. Meanwhile, the selective enrichment of glycopeptides was also investigated from the digests of the mixture containing ASF and MYO (ASF:MYO = 1:10). Considering the effect of functional group density, MSP@PNAMAm-1 and MSP@PNAMAm-4, which had 1.49 and 0.77 mmol/g of alkoxyamine groups, respectively, were applied in glycopeptides enrichment, and the results were shown in Figure 8. Before enrichment, the dominant peaks in the spectrum were due to non-glycopeptides. After enrichment with the two samples, all the dominant peaks (\(m/z\) values of 1627.6, 1755.7, 1950.8 and 3017.4) were attributed to deployeptides from the digest of ASF. What is more, it was interesting that the glycopeptide distribution in the mass spectrum were affected by the enrichment materials. For the MSP@PNAMAm-4, the strongest peak was at 3017.4 (\(m/z\)), while the intensity of this peak was weaker than those at 1627.6 (\(m/z\)) and 1755.7 (\(m/z\)) with MSP@PNAMAm-1. In addition, it was observed when non-glycopeptide was mixed with glycopeptides in a high molar ratio (100:1) (Figure S8, Supplementary Material). The early reports found the same phenomenon [29; 42]. In consideration of the complex conjugation-elution process, it is difficult to explain this phenomenon now, but it is sure that different surface structures have varying interaction with glycopeptides, and the difference might affect the enrichment results. The enrichment ability of MSP@PNAMAm-1 and MSP@PNAMAm-4 were further investigated by profiling the \(N\)-glycoproteinome of a normal human serum sample. We found that the result of MSP@PNAMAm-1 was not good. In contrast, MSP@PNAMAm-4 with the low functional group  
Figure 8: MALDI-TOF mass spectra of the tryptic digest mixture of ASF (asialofetuin) and MYO (myoglobin), the mole ratio of ASF:MYO = 1:10. (**a**) direct analysis; (**b**) analysis after enrichment by MSP@NAMAm-1 and deglycosylation by PNGase F; (**c**) analysis after enrichment by MSP@NAMAm-4 and deglycosylation by PNGase F. The symbols * denote the deglycosylated peptides. Figure 7: SDS–PAGE analysis of the model glycoprotein proteins before and after treatment with MSP@PNAMAm-1 core-shell microspheres.