Comprehensive Multidimensional Separations of Peptides Using Nano-Liquid Chromatography Coupled with Micro Free Flow Electrophoresis

Abstract

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in μFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC × μFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC × μFFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC × μFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC × LC.

Fraction transfer process in on‐line comprehensive two‐dimensional liquid‐phase separations

“…One of the most promising approaches to overcome this issue introduced in recent years has been published by Bowser et al. . The authors used nano‐LC directly coupled to micro free‐flow electrophoresis, which performed continuous separation of the LC effluent.…”

“…The peak capacity achievable in 2-D LC × CE separations is limited by the undersampling of D1 separation during the fraction transfer process. One of the most promising approaches to overcome this issue introduced in recent years has been published by Bowser et al [108,109]. The authors used nano-LC directly coupled to micro free-flow electrophoresis, which performed continuous separation of the LC effluent.…”

Recent developments in capillary and microchip electroseparations of peptides (2013–middle 2015)

“…Various FFE modes, particularly the FF‐ZE and FF‐IEF have been used for prefractionation of complex peptide/protein mixtures in proteomic studies of cells and body fluids . FFE systems have been designed and applied to separation of peptides and proteins also in the microchip formats . However, due to their limited preparative capacity they are more suitable for continuous monitoring of selected analytes than for really preparative purposes.…”

Advances in steady-state continuous-flow purification by small-scale free-flow electrophoresis

“…Early [77][78][79][80] and modern [81][82][83] original research papers and reviews [84][85][86][87] describe a large amount of work devoted to the separation of cells, vesicles, organelles and bacteria.…”