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 ml-1. Both infusion and 20 ml-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-

biotin. Phosphate buffer and ammonium acetate/ammonium formate give a factor 10 x and 100 x less background response, respectively. As regards to sensitivity, ammonium acetate and ammonium formate gave the highest response for biotin and fluorescein-biotin. Consequently, all stock solutions were prepared in methanol (biotin/fluorescein-biotin) or ammonium formate (10 mmol L-1; pH 7.5, protein).

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 L-1; pH 7.5) and ammonium formate (10 mmol L-1; 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 mL-1, 1 ng mL-1) and 20 mL 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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