High efficient large scale proteome analysis strategies with serially coupled long microcolumn

Abstract

Comprehensive analysis of whole proteomes is an extraordinary challenge. LC/MS/MS is commonly used to separate peptides digested from proteomes extracted from cells or tissues. To increase protein identification capacity, improvement on separation efficiency and peak capacity of HPLC and sample fractionation to reduce sample complexity are two effective ways. Increasing column length is one of the effective solutions to improve the separation capacity of &#61549;RPLC. However, it was hard to prepare a long microcolumn due to high backpressure generated during packing procedure. In our recent work, through connecting microcolumns of 5, 10 and 15 cm length via unions with minimal dead volume, long microcolumns with lengths up to 30 cm were obtained, and applied for analyzing protein digests. With a 30 cm long microcolumn (300 &#61549;m i.d.), 318 proteins extracted from E. Coli were identified by &#61549;RPLC-ESI MS/MS (LCQ), compared with 70 proteins by a 5 cm long microcolumn. In addition, such a column was applied in 2-D SCX-RPLC/MS/MS for peptides separation and protein identification. Compared to that obtained with normal 10 cm-long &#61549;RPLC column, higher peak capacity (2365 vs 2015) and more identified proteins or protein groups (1836 vs 1358) with FDR<1% were obtained within shorter time (26.7 h vs 39 h). Furthermore, protein pre-fractionation by preparative microscale solution isoelectric focusing (PMSIEF), peptide separation by &#61549;RPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS, were combined, and applied for high resolution separation and high sensitive detection of proteins extracted from E. coli. By comparison with that identified only by &#61549;RPLC-MS/MS with serially coupled long microcolumn, the identified protein number was increased by 3.53 times (835/236), and the Mws and pIs ranges were extended (2563.0 Da to 219155.7 Da vs 2563.0 Da to 163837.0, 3.98 to 11.81 vs 4.01 to 11.42). In addition, a larger scale proteome analysis by combining PMSIEF fractionation technology with 2-D SCX-RPLC/MS/MS with a serially coupled long &#61549;RPLC column was performed, and more proteins were identified