Au chip

MALDI-TOF MS (ProteinChip Biology System II-c, PBS II-c) was purchased from Ciphergen Biosystems company (Austin, TX, USA), and Au chip was purchased from BioRad Laboratories, Inc. (Hercules, CA, USA). MB-WCX were purchased from Bruker Daltonics company (USA). Sodium acetate, urea, dithiothreitol (DTT), acetonitrile (ACN), Tris-HCl (pH9.0), Trifluoroacetic acid (TFA), sinapinic acid (SPA), water (HPLC grade), 3-[(3-choleamidopropyl) dimethylamino]-1-propanesulfonate (CHAPS), and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)were purchased from Sigma Aldrich (USA).

Eluent (5 µL) was pipetted into another PCR tube and thoroughly mixed with 5 µL of SPA (50% ACN, 0.5% TFA). Next, 1 µL of the mixture was pipetted onto the Au chip (Bio Rad, USA) and left to dry naturally at room temperature. The prepared Au chip was immediately tested by MALDI-TOF MS in the positive-ion mode. The mass spectrometry parameter settings were as follows: laser intensity: 180; detection sensitivity: 8; optimized range of the relative molecular mass: 2,000–10,000 Da; and maximum relative molecular mass: 50,000 Da. Each point on the chip was collected 80 times. Instrument external calibration was performed by standard procedures using the all-in-one peptide molecular mass standards before the MS analysis, which contained 5 polypeptides [i.e., arginine 8-vasopressin (1,084.247 Da), somatostatin (1,637.903 Da), bovine insulin B chain (3,495.941 Da), human insulin (5,807.653 Da), and hirudin (7,033.614 Da)], with the protein molecular weight error <0.1%. To assess the stability and repeatability of the MALDI-TOF MS, 8 within-run analyses were performed using quality control samples (collected from 10 EH patients) during the experiment, to ensure a coefficient of variation of mean peak intensity of quality control protein <16%.