MALDI-TOF Protein Identification Protocol

Three biological experiments were carried out with three technical replicates. The total number of samples analyzed by MALDI was 300. The number of technical replicates for protein identification by MALDI mass spectrometry was 2-3 (up to 5 for important and low-abundance proteins). Individual protein spots, selected on the basis of image-analysis output, were excised and digested in-gel with trypsin (Trypsin V511, Promega, Madison, WI, USA) as previously described [19 (link)]. For MALDI-TOF identification, 0.5–1 μL of the sample (50% solution of acetonitrile in water, 0.1% TFA) was placed on a ground steel MALDI target plate or AnchorChip or SmallAnchor (depending on the protein quantity), and 0.5–1 μL of the matrix (α-cyano-4-hydroxycinnamic acid) (Bruker Daltonics, Bremen, Germany) was added.