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Two‐stage Off‐Gel™ isoelectric focusing: Protein followed by peptide fractionation and application to proteome analysis of human plasma
Author(s) -
Heller Manfred,
Michel Philippe E.,
Morier Patrick,
Crettaz David,
Wenz Christian,
Tissot JeanDaniel,
Reymond Frédéric,
Rossier Joel S.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410106
Subject(s) - fractionation , chromatography , isoelectric focusing , peptide , chemistry , proteome , blood proteins , albumin , haptoglobin , isoelectric point , gel electrophoresis , biochemistry , biology , immunology , enzyme
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 15×15 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 α‐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 p I ‐based separation of peptides appears to be nonuniform based on the theoretically determined p I values of identified peptides. This observation specifically accounts for the neutral zone (p I 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 p I of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.