title: Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.
abstract: The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics.
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Lohnes
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Quebbemann
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Fred
authors.initials: F
authors.lastname: Kobzeff
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; DOE/UCLA Institute of Genomics and Proteomics and UCLA Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: JLoo@chem.ucla.edu.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; DOE/UCLA Institute of Genomics and Proteomics and UCLA Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: RLoo@mednet.ucla.edu.
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D008970:Molecular Weight; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D020033:Protein Isoforms
keywords: Gel electrophoresis; Isoelectric focusing (IEF); Mass spectrometry; Matrix-assisted laser desorption/ionization (MALDI); Proteomics; Virtual 2D gel
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