A Molecular Scanner To Automate Proteomic Research and To Display Proteome Images

Abstract

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annotated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electrotransferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

“…However, the chemicals are very expensive, and scanning has to be made by the use of an expensive fluorescence imager. A molecular scanner to automate proteomic research for the display of proteome images was designed [37].…”

Accuracy of two-dimensional electrophoresis for target discovery in human colorectal cancer

“…The introduction of immobilized or narrow-pH gradients and the combination with mass spectrometry 40 have transformed 2-D PAGE into a powerful analysis tool. However, 2-D PAGE will remain hampered by intrinsic problems.…”

“…Current methods for high resolution 2-D PAGE are capable of separating about 3000 protein spots in one procedure, 41 out of perhaps 500 000 proteins/isoforms present in a human cell. 40 Thus, 2-D PAGE allows only the analysis of a small subset of proteins. Moreover, this subset is biased by the over-representation of abundant proteins, of hydrophilic proteins, and by the relative absence of membrane proteins.…”

Effect of Zinc and DHA on Expression Levels and Post-Translational Modifications of Histones H3 and H4 in Human Neuronal Cells

“…Although proteomics as an entity is relatively new, the methodological and theoretical foundations have been under development for more than three decades (Campostrini, Pascali et al 2004). Two-dimensional protein electrophoresis, coupled with peptide mass fingerprinting analysis by mass spectrometry (MS), has become the most powerful techniques for proteome analysis (Binz, Muller et al 1999). …”