The Gly-X™ N-Glycan Rapid Release and Labeling with InstantPC™ kit utilizes a novel in-solution enzymatic protein deglycosylation followed by rapid labeling of released N-glycans with InstantPC dye. After a simple clean-up step, the glycan samples are ready for analysis by UHPLC, LC-MS, MS/MS and other methods. With deglycosylation and labeling carried out in solution, the method is simple, rapid and suitable for automation. The InstantPC dye delivers unmatched fluorescent brightness and MS signal, which enables a single labeling method to be deployed across different glycan analysis workflows. Other benefits include:
Equipment and reagents provided by user:
Size: 1 kit (96-ct)
Product Code: GX96-IPC
Q. What is the most common adduct ion seen in MS analysis of InstantPC-labeled glycans?
A. In positive mode MS, most biantennary InstantPC-N-glycans will give [M+2H]+2, larger sialylated will be majority [M+3H]+3.
Q. Do you have a list of masses for adduct ions of labeled N-glycans seen by MS in positive and negative mode?
A. Yes, we have a downloadable Excel file listing masses of adduct ions of unlabeled and labeled glycans. The positive mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and InstantPC. The negative mode tab shows glycans labeled by 2-AB, InstantAB, 2-AA, Procainamide and APTS.
Q. You suggest 100 mM ammonium formate for the 5-minute HILIC separation, 50 mM ammonium formate for the 60-minute separations. Is this correct?
A. For fast separations we stick with 100mM buffer, for MS in conjunction with longer separations we use 50mM ammonium formate.
Q. Why is PBS not recommended as a diluent for glycoprotein samples?
A. PBS is compatible with Gly-X InstantPC kit, however, use of PBS for sample dilution may result in artifact peaks. These peaks elute early in the separation, near the free dye peak and should not interfere with N-glycan analysis. For optimal performance, dilution of samples with water is preferred.
Q. Can I use more than the recommended upper limit of 40 µg protein per reaction?
A. Maybe, although it will depend on the protein. You would have to test to ensure that labeled glycans from > 40 µg of your protein can be successfully loaded onto the Gly-X Cleanup Matrix without blockage.
Q. My samples are not loading completely onto the Gly-X Cleanup Matrix (Load Step 9, page 9). What is happening?
A. This may be caused by the nature of your protein sample, or by matrix effects caused by the composition of your formulation buffer. This can be addressed by using less protein per reaction, or by buffer exchanging your protein prior to starting the Gly-X protocol.
Q. Why can I not use ACN to dilute the samples prior to HILIC injection? And can I use something other than DMF for the dilution?
A. If the InstantPC-labeled glycans in Gly-X Eluent are diluted with ACN alone, this may cause the sialylated glycans to precipitate. We have not tested alternatives to DMF for sample dilution.
Product Safety Documentation for GX96-IPC:
|WS0343||Gly-X N-Glycanase (PNGase F)|
|WS0344||Gly-X Digestion Buffer|
|WS0339||InstantPC Dye Solvent|
This kit contains multiple modules, each with their own Certificate of Analysis, which may be searched for & accessed here.