Two-Dimensional Microcolumn Separation Platform for Proteomics Consisting of On-Line Coupled Capillary Isoelectric Focusing and Capillary Electrochromatography. 1. Evaluation of the Capillary-Based Two-Dimensional Platform with Proteins, Peptides, and Human Serum

Abstract

In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.

Recent developments in CE and CEC of peptides

“…Human transferrin could be detected with acceptable accuracy and repeatability at physiological concentration levels (0.5-1.2 mg/mL). A 2-D microcolumn platform consisting of online hyphenated CIEF in hydroxypropylcellulose coated capillaries and neutral stearyl-acrylate (C17) monolithic CEC has been used for separation of model mixtures of 15 peptides, 15 proteins, and albumin-depleted human serum [115].…”

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

“…Developing a reliable and robust interface is a key issue to construct an efficient 2D-CE separation system. Over the past years, a variety of 2D-CE interfaces have been introduced, including cross interface [6], valve [7], microdialysis device [8][9][10] and whole etched porous interface [11]. Dovichi's group [6] first reported a system for automated protein analysis using an interface that aligned two separating capillaries and two waste capillaries which were held in place by tightening the ferrules in the Valco cross.…”

“…Dovichi's group [6] first reported a system for automated protein analysis using an interface that aligned two separating capillaries and two waste capillaries which were held in place by tightening the ferrules in the Valco cross. Rassi's group [7] coupled capillary isoelectric focusing (CIEF) to capillary electrochromatography via a nanoinjector valve to perform 2D separation. Mohan et al [8] employed microdialysis junction as the interface for on-line coupling of CIEF with transient capillary isotachophoresis/zone electrophoresis (CITP-CZE) for the separation of tryptic digests of proteins.…”

“…CIEF is preferably used as the first dimension in the construction of 2D-CE systems [7][8][9][10][11], due to its high separation resolution, high peak capacity, and excellent concentration capability. Carrier ampholytes (CAs) are indispensable to establish a stable pH gradient and usually reduce UV detection sensitivity due to the high absorbance at low wavelengths.…”

Online preconcentration and two‐dimensional separation of cationic compounds via hyphenation of capillary zone electrophoresis with cyclodextrin‐modified micellar electrokinetic capillary chromatography

“…Sahlin reported a CZE-MEKC method for the separation of peptides in a system based on tangentially connected capillaries [15]. Zhang and El Rassi used a commercial microinjectors for connecting CIEF and CEC for the separation of proteomic mixtures [16]. Anouti et al developed heart-cutting 2-D CE in a single capillary for online purification and separation of derivatized amino acids [17].…”