Development Of New Methods For Identification Of Sulfated/Phosphorylated Glycans On Intact Glycopeptides

N-glycosylation of proteins is one of the most common post-translational modifications of proteins and plays important roles in various biological processes,such as cell adhesion,recognition,signal transduction and immunity.However,the glycan itself can also be modified by a variety of functional groups,of which negatively charged phosphate groups and sulfate groups are the most common two.Sulfation generally occurs on complex N-glycans and rarely on high-mannose and hybrid N-glycans.Sulfate groups are often located at C-3 of galactose,C-6 of N-acetylglucosamine,C-4 of N-acetylgalactosamine,and C-3 or C-6 of galactose-N-acetylglucosamine.Glycan sulfation plays important roles in biological processes such as cell adhesion,molecular recognition,control of targeted migration of cancer cells and inflammatory and immune.Moreover,sulfated N-glycan on Ig G has been widely studied as a biomarker of rheumatoid arthritis.The phosphate group generally occur at the C6 position of mannose in high-mannose N-glycan structures,so it is called mannose-6-phosphate（M6P）.M6P glycoproteins are mostly localized in lysosome and defined as the lysosomal hydrolase,which hydrolyze various compounds.However,both the absence of hydrolases and the abnormality of the M6P-dependent pathway can lead to the development of lysosomal storage diseases.The growing maturity of glycoproteomics has greatly facilitated the development of methods for the identification of phosphorylated and sulfated glycans.However,the abundance of phosphorylated and sulfated N-glycans in samples of mass spectrometry is much lower than that of peptides,and the presence of negatively charged peptides can also interfere with the identification signals of these two glycans.In order to identify these two glycans,an enrichment method with high specificity is needed.Meanwhile,the identification of these two negatively charged glycans depends on the accurate identification of their feature ions,which requires specific fragmentation energies.With a combination of optimized fragmentation energies,identifying glycan sulfation and phosphorylation simultaneously can be achieved.In this work,we first used a strong anion exchange column（MAX）to enrich the glycopeptides,which excluded the influence of phosphorylated peptides,and then applied Fe³⁺-IMAC（ferric ions in immobilized metal affinity chromatography）to specifically enrich the sulfated/phosphorylated glycans by the charge interaction.A total of 778 sulfated glycopeptides and 250 sulfated glycoproteins were identified by the new enrichment approach.Compared with previous studies,the number of identifications was increased by3 times and 2 times,respectively.At the same time,our method identified a total of 271glycopeptides with phosphorylated glycans and 111 glycoproteins,which were about two times higher than previous studies.These results indicated that the new enrichment method can indeed increase the number of identifications of phosphorylated,sulfated glycopeptides and proteins,showing the superiority of the new enrichment strategy.In the identification and analysis of sulfated and phosphorylated glycopeptides,we found that the results obtained by the new enrichment method intersected less with those obtained by the traditional enrichment method,showing the different enrichment characteristics of the two methods for sulfated and phosphorylated glycopeptides.And the combination of the two methods could achieve complementary strengths.A total of 981 sulfated glycopeptides,333 sulfated glycoproteins,317 M6P glycopeptides and 137 M6P glycoproteins were identified by the two methods,which maximized the amount of sulfated and phosphorylated glycopeptides and glycoproteins.Besides,phosphorylated and sulfated glycopeptides were fragmented using the fragmentation energy of HCD33%,20%,and 10%in MS2.Among them,HCD33%and 20%could well identify phosphorylated glycopeptides,while HCD33%,20%and 10%gave good identification of sulfated glycopeptides.In summary,this study established approach for large-scale simultaneous identification phosphorylated,sulfated glycopeptides,and profiled the expression of phosphorylated,sulfated glycopeptides and proteins in mouse brain tissue,which provides a data base and technique support for subsequent functional and pathological studies of phosphorylated and sulfated glycoproteins.