Test standard

To prepare the matrix solution, the matrix substance, purified with α-cyano-4-hydroxy-cinnamic acid (HCCA; Bruker Daltonics, Bremen, Germany), was dissolved in the standard solvent consisting of acetonitrile 50%, water 47.5% and trifluoroacetic acid 2.5%. The resulting concentration was 10 mg HCCA/mL. Recombinant human insulin (Sigma-Aldrich, Taufkirchen, Germany) in HCCA solution was added to serve as an internal calibrant for spectrum evaluation. The final concentration of human insulin in 50% aqueous acetonitrile was 10 pg/μL. The exact determination of the insulin peak mass was carried out experimentally by mixing with the Bruker Test Standard and consecutive recording of mass spectra. The insulin peak was detected at an m/z = 5,806.1. The insulin peak functioned as an internal calibrant for all C. coli mass spectra. Insulin proved to be particularly suited, because its mass did not coincide with other recorded biomarker masses. The use of an internal calibrant significantly increases precision in the determination of biomarker mass changes. With this approach, we were able to detect mass differences with a standard deviation of less than 1 Da.