**3. A case study: application of the Fbs1-GYR enrichment method to study N-glycan heterogeneity in Wnt signaling**

The Wnt signaling pathway plays important roles in normal development and in cancer progression [51]. Several enzymes (such as DPAGT1) which are involved in N-glycan biogenesis are regulated by Wnt3a ligand stimulation [52, 53]. Therefore, an N-glycosylation study was performed to reveal potential biomarkers for the detection of Wnt-related cancers. We applied Fbs1-GYR enrichment technology to investigate whether protein N-glycosylation heterogeneity changes upon Wnt3a stimulation in mammalian cells.

Murine recombinant Wnt3a ligand and the Wnt Protein Stabilizer (AMS. bWps) (ASMBio, Cambridge, MA) in combination were able to stimulate canonical Wnt signaling in HEK293 SuperTopFlash STF cells (ATCC CRL-3249) in serum free media. Controls cells (non-Wnt3a stimulated cells) were treated in the same manner but without addition of Wnt3a. A 103.6 ± 3.92 (n = 3) fold change in TopFlash reporter gene expression was observed after 24-hour stimulation with 50 ng/ml Wnt3a and 50 μg/ml of Stabilizer. Note that serum free media was necessary to prevent possible glycoprotein contamination from bovine serum.

Control cells or Wnt3a-stimulated cells were harvested, and total protein was digested with trypsin. N-glycopeptides were enriched with 50 μg Fbs1-GYR purified protein using the N-glyco-FASP method [32] from 200 μg of tryptic peptides prepared from either control cells or Wnt3a-stimulated cells. The enriched N-glycopeptide samples were subjected to LC–MS/MS analysis and N-glycopeptide searching by Byonic as described [37]. 1556 and 1233 N-glycopeptide spectrum matches (N-glyco PSM) were obtained from Wnt3a-stimulated cells and control cells, respectively. (The complete dataset is available upon request from the corresponding author). The numbers of peptide spectrum matches (PSM) are suggestive of the relative abundance of the peptides [26]. Thus, the value of N-glyco PSM is used to evaluate and compare protein N-glycosylation in the Wnt3a-stimulated cells and the control cells. Using criteria of at least a two-fold change and a minimum 10 PSM difference between Wnt3a-stimulated and control cells, 17 proteins were identified exhibiting significant changes in N-glycosylation (**Table 2**). Among them, N-glycosylation of 11 proteins (MPRI, AN32B, PON2, SAP, NOMO3, TMED4, FKBP9, ATRN1, LMNB2, ZMAT4, and BASI) showed a significant increase upon Wnt3a stimulation, while N-glycosylation of 6 proteins (MA2B1, PLOD1, MPRD, MOMO1, HEAT1, and ABCAD) was significantly reduced with Wnt3a stimulation. MPRI (Cation-independent M6P receptor) and MPRD (Cation-dependent M6P receptor) are both mannose-6-phosphate (M6P) receptors. However, they display an opposite response to Wnt3a stimulation with regard to N-glycosylation (**Table 2**, **Table 3** highlighted within the dark blue box). The detected N-glycosylation of


**Table 2.**

*List of 17 proteins with significant differences in overall N-glycosylation upon Wnt3a stimulation. N-glycosylation is evaluated by spectral counting label-free quantification. The scoring criteria was a minimum of 10 PSM difference and a fold change minimum of 2 between Wnt3a-stimulated cells and the control cells.*


#### **Table 3.**

*Comparison of detected N-glycosylation in MA2B1, MPRI, MPRD, and HYOU1 in Wnt3a-stimulated cells and the control cells. The light green rows indicate N-glycoprotein identity. Beneath the protein identity row, N-glycosites are listed in light blue. Beneath each N-glycosite, the respective N-glycan composition is listed. N@ indicates the asparagine with N-glycan modification. PSM numbers of individual N-glycosylation modifications are listed in columns on the right side.*

MPRI increases 11.7 fold, while N-glycosylation of MPRD decreases approximately 3-fold after Wnt3a stimulation. The observed N-glycosylation changes may be due to the changes of protein expression level, which deserves further investigation.

The N-glyco PSM of lysosomal alpha-mannosidase (MA2B1(O00754)), is greatly reduced (5-fold) with Wnt3a stimulation. Interestingly, the N-glycosylation change is mainly due to differential glycosylation of N133 of MA2B1(O00754). **Table 3** shows 11 PSM with a fucosylated N-glycan (HexNAc(2)Hex(3)Fuc(1)) were found at position N133 attached to this mannosidase in control cells, but only one PSM was detected in the Wnt3a-stimulated cells (highlighted in the red box). Thus, we speculate that reduced N-glycosylation may affect stability of this mannosidase in the lysosome resulting in altered N-glycosylation of substrate proteins. The extent of N-glycosylation of some enzymes did not differ significantly between Wnt3a-stimulated cells and the control cells. However, modification of a specific N-glycosite did differ significantly. For example, there was no significant fold change with regard to the total numbers of N-glyco PSM of HYOU1, Hypoxia upregulated protein 1, which were 71 and 119 in control and Wnt3a-stimulated cells, respectively (**Table 3**). However, a 3-fold increase in N-glycosylation at position N931 of HYOU1 was found upon Wnt3a stimulation (57 PSM in Wnt3a-stimulated cells vs. 19 PSM in control cells, **Table 3**, highlighted in the green box). Overall, this study demonstrates that the Fbs1-GYR enrichment method allows for the examination of glycosite heterogeneity of individual cellular proteins, and this study has revealed candidate biomarkers for Wnt-related cancers.
