Optimization of MS Conditions

Different organic and inorganic buffers, such as ammonium acetate, ammonium formate, HEPES, Gly-Gly, and triethanolamine, were selected to study the response of biotin and fluorescein-biotin in MS and compared to phosphate buffer. Biotin and fluorescein-biotin were dissolved in the carrier solution compositions of buffer (10 mM pH 7.5)/methanol (50 50, v/v) at concentrations of 10 ng pl k Both infusion and 20 pl-loop injection experiments were performed with detection by MS in full-scan and SIM mode. Main optimization criteria are the maximum response of biotin and fluorescein-biotin with lowest interference of the carrier solution. HEPES, Gly-Gly, and triethanolamine give very high background response, which significantly hampers the detection of biotin and fluorescein- 

In order to select a carrier solution composition which would provide an overall maximum response for MS detection, two modifiers were selected, acetonitrile and methanol, and two buffers, i.e. ammonium acetate (10 mmol pH 7.5) and ammonium formate (10 mmol L pH 7.5). Biotin and fluorescein-biotin were dissolved in various binding buffer-organic solvent mixtures ranging from 90 10 (v/v) to 50 50 (v/v) at two concentration levels (0.01 ng 1 ng pL ) and 20 pL were injected and analyzed by MS in full-scan and SIM mode. The maximum response was found with 50% methanol, which was about a factor 2x higher than for 10% methanol. Since the proteins can denaturate or protein-ligand complexes can dissociate at relatively low percentages of organic modifier in further experiments only 10% methanol is used in the carrier solution. 

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