P57-T Development of an Automated RP/RP 2D NanoLC/MS Method for Proteomic Analysis

An automated RP/RP 2D nanoLC/MS method was developed and applied for E. coli digest analysis. Even though the first and second dimensions were both carried out with reversed phase (RP), the separation was performed with a high degree of orthogonality due to a sharp difference in the pH of the mobile phases used for each dimension (10 and 2.6, respectively). The automation of the 2D system was realized with the use of a novel on-line organic dilution method: when an organic-containing fraction was eluted from the RP column of the first dimension with pump 1, an aqueous flow was pumped into the system with pump 2 and mixed with the eluted fraction to reduce its organic content. When the fraction was collected with a RP trap column interfacing the two RP dimensions, the organic concentration in the fraction was lowered enough to allow the peptides present in the fraction to be retained. Then a nano-flow gradient was delivered from pump 2 through the trap column and the RP column of the second dimension for further separation. The developed 2D method was successfully applied to sequencing a 500-ng E. coli global digest. The sample was fractionated 30 times with the first dimension. Each of the fractions was run with the second dimension using a 30-min gradient. There were more than 637 proteins identified with a false-positive rate of 3.8%. There were 5171 peptides corresponding to the identified E. coli peptides, of which only about 10% were observed from two or more fractions, indicating a high fractionation quality. These data suggest that the RP/RP method may be preferable to the conventional SCX/RP for proteomic analysis.