Partial phosphorylation has been reported in the regulation of transport process. For example, the phosphorylation degree of tau protein regulates its axonal transport by controlling its binding to kinesin ; dysregulation of tau phosphorylation is observed in Alzheimer��s disease ; ICG-001 cyclin-dependent kinase 5 can increase the phosphorylation degree of perikaryal neurofilament and inhibits neurofilament axonal transport in response to oxidative stress ; and the phosphorylation degree of the nuclear transport machinery has been reported to negatively Carfilzomib regulate entire nuclear transport pathways for the global control of cellular activities. The systematic study of partial phosphorylation will deepen the understanding of its roles in other biological processes. The role of partial N-glycosylation in glycoproteins has seldom been studied because most current glycoprotein analysis methods are incapable of distinguishing partially from fully glycosylated sites. In this study, by comparing glycopeptides with unmodified peptides identified by the ERLIC04 method, the corresponding unmodified peptides were identified for 60 unique glycopeptides in 53 glycoproteins. As shown in Figure 5A, the glycopeptide of GVVDSDDLPLgNVSR eluted 8 or 9 fractions later than its unmodified form, a significant shift for so acidic a peptide. This is evidence of the degree to which the negative charge from sialic acid residues prolongs retention in ERLIC. The glycosylation level at this site in vivo was estimated to be about 1.2% using the peak intensity of the XICs of the glycopeptide and its unmodified form, indicating that our strategy is very sensitive in detecting partial glycosylation. The MS/MS spectra of the peptide of GVVDSDDLPLgNVSR and its unmodified form are shown in Figure 5B and 5C, respectively. In the MS/MS spectrum, its glycosylation site is sandwiched between fragments with the deamidated asparagine, i.e. b12, b13, y6 and y8-y12, the mass of which was about 0.98 Da higher than that of the corresponding b or y ions detected in the MS/MS spectrum of the unmodified peptides. This further validates the assignment of the glycosylation site. The glycoproteins with partially glycosylated sites were also categorized according to their subcellular locations and biological processes using Amigo Go Slimmer. As shown in Figure 5D and 5E, partial glycosylation can occur on proteins in all of the subcellular locations and biological processes except for RNA metabolism and transcription. However, when compared with the distribution of glycoproteins listed in Table 1, partial glycosylation tends to happen on cytoskeleton, nucleus and other cytoplasmic organelles including endosome, lysosome and vacuole, and is also prone to occur on proteins involved in the biological process of cellular component organization and transport.