Capillary Isoelectric Focusing-Based Multidimensional Concentration/Separation Platform for Proteome Analysis

Abstract

An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures. Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240. In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography. The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins. The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg. This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques. The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis. By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

“…2, the degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RPLC separations [50][51][52][53] for the analysis of the same mitochondrial sample. As observed in our previous studies [50][51][52][53], the pI of basic peptides may not be well defined and the basic peptides do not focus as tightly as those of acidic counterparts during the CIEP separation.…”

“…2, the degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RPLC separations [50][51][52][53] for the analysis of the same mitochondrial sample. As observed in our previous studies [50][51][52][53], the pI of basic peptides may not be well defined and the basic peptides do not focus as tightly as those of acidic counterparts during the CIEP separation. In comparison with the shotgun-based multidimensional LC system [54,55], a high degree of peptide overlapping was typically observed with 40-80% of carry over peptides that were identified in the previous salt steps in the first dimension of strong-cation-exchange chromatography.…”

Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation

“…The tryptic digest of a four-protein mixture has been considered as a test sample. IEF fractionation by OGE has been performed on a wide pH range (3)(4)(5)(6)(7)(8)(9)(10), and different experimental conditions to perform CE have been tested. Also, to know whether CZE can really be considered as a method complementary to OGE, some significant OGE fractions have also been analyzed by RP-HPLC.…”

Micro-proteome analysis using micro-chromatofocusing in intact protein separations

“…A number of methods have been developed to fractionate the protein content of a cell on a micro-proteomic scale [5,6,7,8]. These methods may use an approach based upon fractionation of intact proteins [6,9] or alternatively fractionation of the protein content of a cell following total digestion of the proteins into peptides [5,10,11,12].…”

“…These methods may use an approach based upon fractionation of intact proteins [6,9] or alternatively fractionation of the protein content of a cell following total digestion of the proteins into peptides [5,10,11,12]. Both of the approaches have been used in previous work for analysis of small amounts of sample.…”