Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two-dimensional electrophoresis and alternatives

Abstract

Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

Gradient chromatofocusing-mass spectrometry: A new technique in protein analysis

“…T here is great interest in developing a mass spectrometry (MS)-compatible high-performance liquid chromatography (HPLC) technology which performs charge-based separations, a principal dimension in the 2D-gel electrophoresis technique, for application in proteomic and protein characterization studies. Development of such a HPLC technique would be progress towards addressing the limitations of the 2D-gel electrophoresis technique, which has been hampered by its design incompatibility for direct interfacing with MS (requiring excision of each protein band from the gel), limitation in quantitative dynamic range, inability of determining small proteins (5-8000 Da), high labor intensity, long sample run times, as well as other disadvantages [1,2].…”

“…Example scanning m/z and deconvoluted spectra are given in Figure 3 for ␤-lactoglobulin B. Experimentally determined detection limits are summarized in Table 1. Experimental molecular masses matched theoretical values within Ϯ0.010 to 0.033% (Ϯ100 to 330 ppm), which is within the reported accuracy of quadrupole techniques (0.01% to 0.04%) [1]. All protein peaks gave scanning m/z spectra that could be deconvoluted to determine correct molecular masses.…”

“…Development of such a HPLC technique would be progress towards addressing the limitations of the 2D-gel electrophoresis technique, which has been hampered by its design incompatibility for direct interfacing with MS (requiring excision of each protein band from the gel), limitation in quantitative dynamic range, inability of determining small proteins (5-8000 Da), high labor intensity, long sample run times, as well as other disadvantages [1,2].…”

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography

“…This approach has already been found to be sufficient to deal with smaller subsets of the proteome (i.e. several hundred proteins) (Hille et al, 2001). These studies also clearly demonstrate that this methodology is not yet suitable for the analysis of a whole proteome due to its enormous complexity.…”

The Human Proteome Organization (HUPO) and Environmental Health

“…Many established proteomic technologies such as 2D gel-MS or multidimensional liquid chromatography-MS currently function extremely well as discovery-based platforms capable of linking gene products to function (Gagnon et al 2002) or localities (Bruno et al 2002) within the cell (Jung et al 2000). Recent technologic advances make possible the identification of hundreds of proteins in a single experiment at high throughput commerical facilities and through the release of new technologies like ICAT (isotope-coded affinity tags) (Gygi et al 1999a) for tandem MS analysis that permit simultaneous detection of high abundance proteins and low copy gene products alike (Hille et al 2001;Honore 2001).…”