Protein Modification Analysis of GM2 Activator Protein Mutants by High Performance nano-LC ESI FT-ICR Mass Spectrometry

Abstract

GM2AP is an 18 kDa protein that is involved in the catabolism of the ganglioside GM2. GM2AP extracts GM2 from intralysosomal vesicles and orients the oligosaccharide head group for hydrolytic cleavage by the hydrolase, HexA. Mutations in GM2AP or HexA lead to an accumulation of GM2 in the lysosomes, causing lysosomal storage diseases such as Tay Sachs or the AB variant of Sandhoff's disease. Our lab is utilizing site directed spin-labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy to probe the conformational changes and the membrane bound orientation of GM2AP. This protein contains eight naturally occurring cysteine (CYS) residues involved in four disulfide bonds. With site directed mutagenesis, a ninth CYS residue is introduced as the reporter site for spin labeling . A series of 10 single CYS mutants have been generated. To validate the EPR results, the mass spectrometry protocol described here was developed to characterize spin-labeled GM2AP samples. For mass spectrometry measurements, either biotin-linked maleimide (BM) or 4-maleimide tempo (4MT) were used to modify and trap available CYS residues in a thioesterbond. The remaining eight native CYS residues, which are disulfide bonded, are then reduced and modified with iodoacetamide. Samples were analyzed by high performance nano-liquid chromatography electrospray ionizaton Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) equipped with a 14.5 T magnet. FT-ICR spectra of enzymatically digested BM-labeled or 4MT-labeled GM2AP were utilized to determine to which CYS residue is modified with the maleimide moeity. Those sites labeled with acetamide are inferred to have been disulfide bonded. The fragment that contains the maleimide moeity tells us, which CYS residue (and how many CYS residues) is accessible for reaction with the spin label for EPR studies.