Iduronate-2-sulfatase proteome isolation from mouse brain and identification of changes in the proteomic profiling in murine model for Hunter syndrome

Iduronate-2-sulfatase (IDS) is a sulfatase that plays a key role in heparan sulfate and dermatan sulfate degradation. Mutations affecting the gene encoding IDS produce the X-linked lysosomal disease mucopolysaccharidosis II (MPSII). This pathology is characterized by intracellular accumulation of both substrates causing severe damage to the central nervous system and other tissues. We have addressed proteomics methodologies to identify the IDS interacting proteins to get insights into its physiological roll in brain. On the other hand, we analyzed the differential complex protein expression in brain via Blue Native (BN) PAGE. Heterologous expression and purification of human IDS. The protein was produced in Pichia pastoris and it was purified by anion exchange and molecular exclusion chromatography. Total protein extracts from mouse brain tissue were prepared following a standard procedure. Isolation of IDS-interacting partners by IDS-sepharose affinity chromatography. An IDS-sepharose affinity chromatography column was built by coupling CNBr-activated with purified human recombinant IDS. A bovine serum albumin (BSA)-Sepharose column was constructed in a similarly and used to clear the brain extracts. Identification of IDS-interacting partners by HPLC-ESI-MS2 and (BN-PAGE). Fractions were eluted from the IDS-affinity column and precipitated by TCA-acetone protocol. The proteins were subjected to in-gel tryptic digestion. BN-PAGE was implemented for the separation and identification of mouse brain protein complex from WT and IDS-KO (024744, The Jackson Laboratory), in an approximate mass range of 10 kDa to 600 kDa, using a specific antibody. Extracted peptides and protein complex from affinity chromatography and BN-PAGE were analyzed by HPLC-ESI-MS2. Mass spectra were analyzed with the database MASCOT search program. Putative proteins analyzed by HPLC coupled with electrospray ionization MS2 were classified into different subcellular localization groups. A differential pattern expression of the complex was observed in liver compared with brain from WT and KO mice.