Anchorchip 384 maldi target plate

The detailed procedure for identifying the discriminative peaks using an UltrafleXtreme (Bruker Daltonics, Bremen, Germany) tandem mass spectrometer has been described previously [18 (link)]. Briefly, before the analyses were conducted, the serum samples were purified using ZipTip C18 tips. The obtained eluates (4 µL) were subjected to nanoLC separation, automatically mixed with HCCA matrix solution, and spotted onto an AnchorChip 384 MALDI target plate (Bruker Daltonics, Bremen, Germany). The experiments were performed in the reflectron positive ion mode of the mass spectrometer in the mass range of m/z 700–3500. Protein–peptide identification was based on the SwissProt database and Mascot 2.4.1 search engine. The database searches were taxonomically restricted to Homo sapiens.

The detailed protocol used for identifying the discriminative peaks using a nanoLC- MALDI-TOF/TOF MS (Bruker Daltonics, Bremen, Germany) platform has been described in our previous paper [33 (link)]. For the identification analysis, we pooled the same samples used for protein–peptide profiling. In brief, the serum samples subjected to analyses were first purified using ZipTip C18 micropipette tips. The obtained eluates in a volume of 4 µL were subjected to nanoLC separation. The obtained fractions were mixed with HCCA matrix solution and automatically spotted onto an AnchorChip 384 MALDI target plate (Bruker Daltonics, Bremen, Germany). The MS and MS/MS experiments were performed in the reflectron positive ion mode in the mass range of m/z 700–3500. Proteomic identification was based on the MS/MS fragmentation spectra, the SwissProt database, and the Mascot 2.4.1 search engine. The database searches were taxonomically restricted to Homo sapiens.