What is the general process of Protein ID by Mass Spectrometry?
It includes the following steps: 1). Gel treatment: Gel is washed multiple times to remove staining dye and other chemicals inhibitory to Mass Spectrometry 2). In-gel trypsin digestion: Trypsin digestion buffer is added and protein is digested in gel at 37°C 3). Peptide extraction: Digested peptide sample is extracted from gel by extraction buffer 4). Desalting: The digested tryptic peptides are desalted by C-18 Zip-tip (Millipore) 5). Spotting: Sample is mixed with matrix buffer and spotted on MALDI plate 6). MALDI-TOF: MS spectra are obtained using ABI4700/ABI4800 proteomic analyzer 7). MALDI-TOF/TOF: Ten to twenty most abundant peptides are further subjected to fragmentation (MS/MS) 8).
