New England Biolabs, P0872S, N-Glycopeptide Binding Protein

Related Categories Glycobiology,, Proteome Analysis Applications Glycobiology & Proteomics,, Glycoprotein Analysis Specification Storage Buffer 20 mM Tris-HCl 50 mM NaCl 0.2 mM TCEP pH 8 Heat Inactivation No Molecular Weight Theoretical: 45 kDa Unit Assay Conditions Functional Testing (N - Glycan Enrichment) - A 200 μL reaction in 20 mM Tris-HCl, 50 mM NaCl, 0.2 mM TCEP, 10% acetonitrile, pH 8.0 containing 1 nmol of fluorescently labeled sialylglycopeptide and 100 μg N - Glycopeptide Binding Protein incubated for 30 minutes at 4°C results in ≥40% enrichment as determined by UV-Vis Spectroscopy at 520 nm. FAQ Q: How does N-Glycopeptide Binding Protein improve glycoproteomic analysis? A: N-glycopeptides typically constitute only a small fraction of the total proteolyzed peptide pool of a complex proteome sample; therefore, N-glycopeptides are often overwhelmed by more abundant non-glycopeptides in an LC-MS experiment. Additionally, the ions with the highest ionization efficiency, which are typically non-glycopeptides, are selected for MS/MS fragmentation to produce glycan compositional information and enable glycopeptide discovery. This can lead to severe underrepresentation of glycopeptides in an LC-MS run. Enrichment by N-Glycopeptide Binding Protein significantly reduces the overall number of non-glycopeptide species in a sample. More MS/MS cycle time will be dedicated to the enriched N-glycopeptides, achieving drastically improved N-glycopeptide spectra match and glycoprotein identification and discovery. Q: What is the specificity of N-Glycopeptide Binding Protein? A: N-Glycopeptide Binding Protein has been engineered to bind N-glycosylated peptides and proteins with a broad variety of N-glycoforms, including high mannose, complex, and hybrid, as well as fucosylated and non-fucosylated, sialylated and non-sialylated, with bi-, tri- and tetra-antennary structures. It also binds to non-mature glycoforms such as paucimannose and those with mannose-6-phosphate. It does not, however, bind N-glycans with bisecting GlcNAc. Q: How strong does the N-Glycopeptide Binding Protein bind to a glycopeptide? A: According to this study: Chen 20171, the Kd between the N-Glycopeptide Binding Protein, and a model sialylglycopeptide is in the micromolar or sub micromolar range. 1. Chen M. et al. (2017). An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides. Nat Commun. May 23;8, 15487. Q: Can N-Glycopeptide Binding Protein bind and enrich free glycans? A: Yes, N-Glycopeptide Binding Protein can bind free glycans if the GlcNAc on the reducing end is in its free form, in which the pyranose and open-ring form are in equilibrium. However, the six-member ring (pyranose) structure is required for binding. Free glycans with an entirely open-ring structure, such as those labeled by reductive amination, will not bind to N-Glycopeptide Binding Protein. Free glycans with instant labels (e.g., Waters GlycoWorks RapiFluor-MS label) will not bind to N-Glycopeptide Binding Protein. An asparagine attached to the free glycan is not required for binding, nor is any peptide sequence. Q: Can N-Glycopeptide Binding Protein bind an intact glycoprotein? A: Yes, N-Glycopeptide Binding Protein can bind intact glycoproteins. However, the binding between the N-Glycopeptide Binding Protein and an intact glycoprotein is impacted by the higher order structure of the protein, the locality, and the structure of the glycans. As a result, although it is possible to enrich intact glycoproteins using this binding protein (in an immobilized format but not in a filtration-based workflow), the effectiveness of pull-down will be glycoprotein dependent. Q: What influences the binding affinity of the N-Glycopeptide Binding Protein? A: In addition to the pyranose glycan structure requirement, several factors can affect the binding affinity of this protein with its substrates. Lower pH will lead to decreased binding; the optimal pH is 7.0 - 8.5. Salt concentration does not appreciably affect the binding. Denaturants and surfactants should be avoided.