Mass Spec Kit
When performing mass spectrometry of large proteins, such as IgG molecules, there is often a need to remove the glycans because of poor quality of the data otherwise obtained. Additionally, fragmentation of IgG can visualaize individual parts of IgG and allow for studies in more detail. The Mass Spec kit contains both the FabRICATOR and IgGZERO enzyme for performing deglycosylation and fragmentation of 1 mg IgG in one hour. Mass spectrometry of samples can then be run directly without further purification.
Effect of deglycosylating IgG Fc
A
B
Mass spectrometry of Fc-fragments from human polyclonal IgG purfied from plasma. (A) 1 mg of gG was incubated with 1000 U of IdeS for 30 minutes and analyzed by mass spectrometry. (B) 1 mg of human IgG was incubated with 1000 Units of IdeS for 30 minutes followed by incubation with 1000 Units of EndoS for 45 minutes and finally analyzed by mass spectrometry. Utilization of RPC18 LC-ESI-MS generated a well-defined protein envelope with a deconvoluted mass of 24137 Da corresponding to the deglycosylated IgG1 heavy chain cleaved at position Gly236.
Homogeneity of F(ab')2 fragments - Pepsin vs FabRICATOR.
A
B
Mass spectrometry (LS/MS) of F(ab’)
2-fragments from humanized monoclonal IgG (Hercptin?). (A) 1mg of IgG was digested using Pepsin (Sigma) for 2 hours pH 3.5. F(ab’)2 fragments was isolated by size exclusion chromatograpy before analyzed by mass spectrometry. (B) mg of IgG was digested using FabRICATOR for 30 minutes andF(ab’)2 fragments was isolated using size exclusion chromatograpybefore analyzed by mass spectrometry.