Msp 96 microscout target

For determination of binding between metal-cations and GRNs, 1 mM metal-cations were incubated with the reduced, metal-free samples of 20 μM GRN-3 (MW 6367.7 Da) or GRN-5 (MW 6017.7 Da) in 20 mM HEPES buffer at pH 7.0 in the presence of 500 μM tris(2-carboxyethyl)phosphine hydrochloride Characterization of the protein-metal complexes was performed on a Bruker Datonics Microflex LT/SH ToF-MS system. For analysis of metal-protein reactions, 95.5 ng of GRN-3 and 90.2 ng of GRN-5 (15 pmol) were spotted separately onto a Bruker MSP 96 MicroScout Target with a 1:1:1 ratio of sample:sinapinic acid matrix(saturated with acetonitrile and water): acetone. Instrument calibration was performed using Bruker Protein Calibration Standard I (Bruker Daltonics). Alkylation reactions were carried out by incubating respective metal-GRN samples prepared as described above, with 1 mM iodoacetamide for 2h at room temperature. The samples were then prepared for analysis using MALDI-ToF-MS by spotting onto a Bruker MSP 96 MicroScout Target with a 1:1:1 ratio of sample:sinapinic acid matrix (saturated with acetonitrile and water): acetone. The MALDI-ToF data were processed and analyzed using the Bruker flexAnalysis software (Bruker Daltonics); the peak assignment and baseline subtraction were performed using the respective tools within the software.

Pellet characterization of the co-incubated protein complexes was performed on a Bruker Datonics Microflex LT/SH ToF-MS system. Samples were prepared by mixing the supernatant or pellet aliquots from respective reactions with 12.33 ng of insulin (used as an internal standard). The pellet samples were dissolved with formic acid in a 1:1 ratio to allow disaggregation of fibrils and then spotted onto a Bruker MSP 96 MicroScout Target with 1:1 ratio of sample:sinapinic acid matrix in saturated acetonitrile and water. The laser intensity was kept constant at 75% with 3.5× detector gain. Instrument calibration was performed using Bruker Protein Calibration Standard I (Bruker Daltonics).