Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher-energy C-trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N-glycosylation sites of glycoproteins. The combination of the very informative collision-induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple-stage tandem mass spectrometry. The resulting MS(3) spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut-off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites.
Children with Neurofibromatosis type 1 (NF1) are increasingly recognized to have high prevalence of social difficulties and autism spectrum disorders (ASD). We demonstrated selective social learning deficit in mice with deletion of a single Nf1 gene (Nf1+/−), along with greater activation of mitogen activated protein kinase pathway in neurons from amygdala and frontal cortex, structures relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate/GABA neurotransmission; deficits in long-term potentiation; and specific disruptions in expression of two proteins associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (ADAM22) and heat shock protein 70 (HSP70), respectively. All of these amygdala disruptions were normalized by co-deletion of p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide novel insights and therapeutic targets for NF1 and ASD patients.
Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins.
The cell surface glycoprotein ␥-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.
The assignment of protein glycosylation sites and their microheterogeneities are of biological importance, yet such characterization is still considered to be analytically very challenging. Several approaches have been recently developed to improve the characterization of glycosylation sites of proteins, including lectin and HILIC enrichment-based methods coupled to mass spectrometry. However, unequivocal assignment of protein glycosylation remains to be a daunting task, prompting continuous demands for the development of sensitive and cutting-edge analytical approaches. beta-N-Acetylglucosaminidase (endo-beta-GlcNAc-ases, Endo-M) is an endoglycosidase capable of hydrolyzing N,N'-diacetylchitobiose moiety in N-linked oligosaccharides bound to the asparagine amino acid residue in various glycoproteins. An attractive feature of this enzyme is its ability to cleave the N,N'-diacetylchitobiose moiety while leaving an N-acetylglucosamine residue bound to the protein. This enzyme is also known to be inactive in the presence of core fucose residue linked to the reducing-end N-acetylglucosamine residue (GlcNAc). Here, we describe an approach capitalizing on these features of Endo-M to (a) determine the glycosylation sites of proteins and the occupancy of these sites, and (b) determine the attachment sites of fucose residue containing N-glycans. The latter is important because of its biological implications. Tryptically digested glycoproteins, which were subjected to Endo-M treatment, were analyzed by LC-MS/MS. Systematic evaluation of the activity of Endo-M toward different glycan structures indicated a dependence of enzyme activity on the complexity of the glycan structures. Efficient release of N-glycans using Endo-M is only achieved through the inclusion of a battery of exoglycosidases to reduce the complexity of the attached glycans and subsequently prompt an effective enzymatic release. Upon Endo-M/exoglycosidase treatment of tryptically digested glycoproteins, glycosylated sites retain GlcNAc residue. The resulting peptides with GlcNAc residues attached to the glycosylation sites are easily assigned through LC-MS/MS analysis and subsequent database searching of the generated tandem MS of such entities. Comparing the LC-MS/MS results of the tryptic digest of glycoproteins treated with PNGase F and Endo-M/exoglycosidases allowed the assignment of core fucose residues to N-glycan reducing-ends. The detection of glycosylation sites only in the tryptic digest of PNGase F treated samples suggested core fucosylation of the attached N-glycans to such sites. This strategy was initially validated using model glycoproteins. It also proved to be useful in determining the glycosylation sites of blood serum glycoproteins.
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