Multidimensional liquid chromatography based integrated platform for proteome study

To achieve high efficiency, high resolution, high accuracy and high throughput analysis of proteomes, an on-line integrated platform with the combination of proteins separation by 2D-SEC-RPLC, removal of organic modifier by an hollow fiber dialysis membrane, protein digestion by immobilized enzymatic reactor (IMER), peptides trapping by parallel C18 trapping columns, peptides separation by RPLC, and protein identification by ESI-MS/MS, was established, and applied for the analysis of proteins extracted from mouse brain. In our protocol, proteins were first separated by SEC with an analytical column. With a time interval of 10 min, eluants were divided into three fractions, and in turn trapped and desalted on a C8 trapping column, followed by separation by RPLC. For each RPLC run, the eluant was further divided into three fractions, with the time interval of 100 min. Before on-line digestion with IMER, each fraction from (RPLC was passed through a hollow fiber membrane based interface (with cut of molecular weight of 3 000), to on-line remove acetonitrile (ACN). To fasten such a procedure, 50 mM NH4 HCO3 containing 5% (v/v) CAN was used as the exchange buffer, with the flow rate of 2 mL/min. Subsequently, proteins could be on-line digested by IMER with trypsin immobilized on hybrid monolith inorganic-organic matrix without stop flow. Finally, peptides were separated and identified by RPLC-ESI-MS/MS. With such a platform, 1 055 proteins could be identified from mouse brain within 18 hr. All these results demonstrate that such an integrated platform might be promising to achieve high throughput analysis of proteomes with improved resolution, accuracy and throughput