Purified recombinant human insulin

For preparation, the α-cyano-4-hydroxy-cinnamic acid (HCCA) matrix purified matrix substance (Bruker Daltonics, Bremen, Germany) was dissolved in standard solvent: acetonitrile 50%, water 47.5% and trifluoroacetic acid 2.5% (Sigma-Aldrich, Taufkirchen, Germany) to a final concentration of 10 mg HCCA/mL. Purified recombinant human insulin (Sigma-Aldrich, Taufkirchen, Germany) was added as internal calibrant to the HCCA-matrix solution. Human insulin was dissolved to a final concentration of 10 pg/μL in 50% aqueous acetonitrile. The exact mass of the insulin peak was determined experimentally by mixing with Bruker Test Standard: m/z = 5806.1. The insulin peak was used for internal calibration of all C. jejuni ssp. jejuni mass spectra because it did not coincide with any other observed biomarker masses. Using an internal calibrant substantially increased the precision when determining variations of biomarker masses. Using this approach, we were able to detect differences in mass of up to 1 Da.