Biomedical Engineering Reference
In-Depth Information
ates a spectrum of the fragments that pass through the detector. Tandem mass
spectroscopy allows dentification of individual compounds such as proteins in
complex mixtures.
After this digression into some methodological background, I now return to
the first of two methods to analyze large-scale protein expression, two-
dimension protein gel electrophoresis (for in-depth reviews, see (6,25)). In this
technique, soluble proteins in a cell are isolated and separated electrophoreti-
cally in two dimensions (hence the name). In the first dimension, proteins are
separated according to their mass. Then the separated proteins are fractionated
along a second dimension according to their charge or, more precisely, their
isolectric point, in a technique called isolectric focusing. After staining proteins
using agents such as silver or Coomassie blue, one is left with a complex pattern
of thousands of spots, each representing one (or more) proteins that are ex-
pressed in the analyzed cells (see Figure 1 for an example). Individual proteins
can then be manually excised from the gel and enzymatically digested into
smaller peptides. (The most commonly used approach here is digestion with the
enzyme trypsin, which cleaves proteins C-terminally of every arginine or ly-
sine). Cleavage products can then be identified through MALDI-TOF mass
spectrometry, either from large protein databases or from the completely se-
quenced genome, if available.
This approach suffers from limitations that can partially be overcome by
automatization. They include the necessity to extract individual protein spots
manually for analysis, and the limited reproducibility of the electrophoretic
separation, especially across different laboratories. Another limitation, less eas-
ily overcome, is the approach's limited dynamic range. It can only detect pro-
teins of moderate to high abundance and resolve "only" on the order of 1,000
proteins per gel. (Compare this with the complexity of the human protein com-
plement of several hundred thousand proteins, if one includes the hundreds of
alternatively spliced variants of many of the 30,000 human genes.) A second
limitation, shared with many other approaches, is that the technique is not ame-
nable to analyze insoluble membrane proteins, which may account for a large
fraction all proteins in a cell.
A second prominent approach to study protein expression on a large scale
circumvents two-dimensional gel electrophoresis. It identifies soluble proteins
directly from complex protein mixtures extracted from living cells and typically
involves three steps (25). The first of them is protein extraction from a cell and
chemical or enzymatic digestion of these proteins into peptides. In a second step,
the chemical complexity of the resulting mixture is reduced, for example,
through separation by capillary electrophoresis. The resulting peptide fractions
can then directly enter the third step: the analysis of identified peptides through
tandem (MS-MS) mass spectrometry. Such direct mass spectrometry of complex
protein mixtures can potentially identify thousands of proteins and, once auto-
mated, is a rapid analysis tool. Identification rates of 10 4 peptide sequences per
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