Two-stage Off-Gel? isoelectric focusing: Protein followed by peptide fractionation and application to proteome analysis of human plasma

Abstract

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.

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

“…Additionally, the pI information has already been found as important data for validating peptide identifications. 17,18 CE as a Subsequent Separation…”

“…For these different reasons, OGE has already been used as a first dimension before LC-MS/MS for proteomic studies. 17,18 In this work, CE has been assessed as a subsequent separation step to OGE for complex peptide mixture separation. The tryptic digest of a four-protein mixture has been considered as a test sample.…”

Recent advances in blood‐related proteomics

“…However, SELDI/ MS is a partial analysis, not giving sequence data in many cases. Finally, other methods, combining different separation techniques (LC, multidimensional chromatography, tryptic digestion and multidimensional chromatography of peptides, off-gel electrophoresis) followed by MS, are currently more and more employed [11][12][13][14][15]. Recently, because a broad range of mass spectrometers have been used, several investigators proposed a new format for MS data, which will hopefully facilitate data management, interpretation, and dissemination of information in proteomic research [16].…”

Porous silica enhanced proteolysis during Off-Gel separation for efficient protein identification

“…Depending on the complexity of sample and desired resolution, a typical OGE takes 6 to 24 h. It can be used for the fractionation of proteins extracted from tissue or cells, and then the peptides digested from each protein fraction [11]. In coupling with capillary zone electrophoresis (CZE), the 2D separation can provide orthogonality comparable with reverse phase chromatography coupled to strong cation exchange chromatography (RP/SCX) [12].…”

“…In coupling with capillary zone electrophoresis (CZE), the 2D separation can provide orthogonality comparable with reverse phase chromatography coupled to strong cation exchange chromatography (RP/SCX) [12]. However, a typical OGE-MS proteome strategy is a tedious process requiring the collection of fractionated proteins, adjusting the system pH of each fraction into preferable condition for trypsin digestion, offline overnight proteolysis, and peptide fractionation [11,13,14]. Sample handling between the steps can lead to sample loss.…”