Protein calibration standard 1 and 2

To prepare the MALDI target, 1 μl eluted sample was spotted onto the MTP target plate (Bruker Daltonics), dried naturally at room temperature, 1 μl CHCA matrix solution (6 mg/ml in 4% trifluoroacetic acid/50% acetonitrile) was absorbed and covered on the dry sample site following by 1:1 ratio dry point method, and then, the target point was air‐dried (cocrystallization). Flexcontrol version 3.4 (Bruker Daltonics, Germany) was used with the following settings: MS scanning range: 1‐10kda, spectral laser intensity: 75%, the MS analysis voltage of ion source 1, and ion source 2 in the positive ion linear mode was 19.6 kV and 18.3 kV, respectively, and the prism voltage was 6.9kV. Prior to formal test analysis, the instrument was calibrated using Protein Calibration Standard I and II (Bruker Daltonics,). All tests were performed in a blind manner. In addition, in order to evaluate the repeatability and overall stability of the mass spectrometer, 30 serum samples from controls were randomly selected and mixed into quality control samples for internal test.

For MALDI-TOF mass spectrometry all samples were purified using C4 ZipTip® pipette tips (Merck) before analysis as described in the supplied manual. The purified sample (1 µl) was mixed with 1 µl of DHAP-matrix (7 mg 2,5-dihydroxyacetophenone, 375 µl ethanol, 125 µl of 16 mg ml⁻¹ di-ammonium hydrogen citrate) and 1 µl of 0.1% TFA and applied onto a metal target plate. The samples were analyzed in linear positive mode (LP_30-210 kDa according to Bruker Daltonics) using an AutoflexTM speed MALDI-TOF/TOF device (Bruker Daltonics). Protein Calibration Standard I and II (Bruker Daltonics) were applied for calibration of the device.