Array based capillary IEF with a whole column image of laser‐induced fluorescence in coupling to capillary RPLC as a comprehensive 2‐D separation system for proteome analysis

Abstract

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.

Comprehensive two‐dimensional separation in coupling of reversed‐phase chromatography with capillary isoelectric focusing followed by MALDI‐MS identification using on‐target digestion for intact protein analysis

“…Based on our previous studies [19,20], the objective of this work was to develop an effective multidimensional fractionation and separation procedure that could be combined with MS to identify proteins within complex mixtures. First, the feasibility of the 2-D liquid-based protein fractionation strategy utilizing cRPLC and coupled to CIEF separation was tested with a mixture of four model proteins with different pIs and molecular masses.…”

“…A 2-D separation system, coupling capillary RP LC (cRPLC) to CIEF, has been developed for protein and peptide mapping by our group [19,20]. RP LC and CIEF are both high-efficiency separation technologies.…”

Modern Biotechnology in China

“…Zhang et al reported the development of PCAS (ProteinCentric Annotation System) as an online resource of pre-computed proteome annotation data; PCAS gives better annotation coverage for model proteomes by employing a wider collection of available algorithms [98] . Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), Mao et al proposed a comprehensive 2D system with laser-induced fluorescence for protein mapping [99] .…”

Capillary electrophoresis‐based separation techniques for the analysis of proteins

“…Because cRPLC has an orthogonal separation mechanism to CIEF, the 2-D system provides high-resolution separation for proteins, with a peak capacity of more than 10 000. Recently, cRPLC (first dimension) coupled to an array CIEF (second dimension) with a novel WCID has been developed for peptide mapping [250]. An array of up to 60 capillaries was assembled in the system, allowing quantitative detection of thousands of focusing protein bands.…”