Integrated proteomics sample pretreatment method based onSCX/SAX mixed packing

An integrated technology for preparing proteomics samples was developed based on SCX/SAX mixed beads. Unlike previously reported SISPROT strategies, SCX/SAX mixed beads were used to replace SCX beads. A mixture of BSA and LYZ was chosen as the model protein to evaluate the protein retention behavior of the SCX/SAX beads at pH of 3, 7.4, and 12. The mass ratio of SCX/SAX was optimized using the BCA method. Further, the protein loss caused by pH replacement during digestion was systematically investigated by the SDS-PAGE method. The results indicated that at a pH of 7.4, the optimized mass ratio of SCX/SAX mixed beads was 1:1, and the loading capacity of SCX/SAX mixed beads with the mass ratio of 1:1 was 180 µg, which is almost twice that of SCX beads. Furthermore, pH replacement during digestion did not affect the protein retention behavior of SCX/SAX mixed beads. The improved SISPROT method was used for processing a limited quantity of colon cancer tumor samples. For comparison, the traditional SISPROT method based on SCX beads was used. The results indicated that the number of protein identification, unique peptides and peptide spectrum matching obtained from the two methods were comparable. These results confirmed the applicability of the method as a proteomics reactor. The method is expected to be a more generic sample preparation technology because it can be operated at physiological pH and exhibits significantly improved loading capacity and reduced sample loss compared with those of the traditional SISPROT method.