Proteome Analysis

An essential step of proteomics is the identification and mapping of the proteins separated by 2D elec-trophoresis. From a mammalian organism, comprising approximately 100 000 possible gene products,

Figure 5 Partial 2D gel images showing soluble proteins of (A) H. influenzae, (B) E. coli and (C) B. subtilis. The proteins were separated as stated in the legend to Figure 1. Note the similarity in the distribution of the major proteins in the three bacterial organisms.

approximately 1000-2000 protein spots can be visualized on one 2D-gel, using Coomassie blue. Higher numbers can be detected, following staining with silver or after radiolabelling. Approximately one-half of the visible spots are available in sufficient quantities to be analysed for identification. Figure 5 shows the analysis by 2D electrophoresis of the proteomes of three bacteria, H. influenzae, Escherichia coli and Bacillus subtilis. The genomes of the three microorganisms have been completely sequenced, so that theoretically all expressed proteins can be mapped. This has however not yet been accomplished. The largest 2D proteome maps, such as that of H. influenzae prepared at F. Hoffmann-La Roche, Basel, include approximately 500 mapped proteins. Many of the unidentified proteins are not expressed in sufficient amounts to be visualized.

For the mapping of proteomes of the various organisms, protein enrichment steps need to be introduced before analysis. We have used several chromatographic steps, such as heparin chromatogra-phy, hydrophobic interaction chromatography, chromatofocusing, hydroxyapatite chromatography and several other approaches, to enrich the low-abundance gene products of H. influenzae and E. coli.

Additional enrichment steps are required for an efficient mapping of proteins present at low abundance, such as cytokines or transcription factors. Figure 6 shows an example of protein enrichment by hydro-phobic interaction chromatography. One protein (enolase), represented by a strong spot in the 2D map of the total protein extract, is highly enriched after chromatography. Another example of protein enrichment, this time using heparin chromato-graphy is shown in Figure 7. In two fractions collected from the column, proteins which are not visible in the 2D gel image of the total extract can also be detected.

On a 2D map, proteins are often represented by more than one spot. Figure 3C shows an example of a brain protein represented by five spots, in two locations, with different pi and Mr values. Presently, we do not know the reasons and the biological significance for most of these cases of observed heterogeneity. It may be the consequence of post-translational modifications, such as deamidation, phosphorylation or glycosylation, which result in the alteration of the pi of the molecule and its focusing position. Another reason may be the carbamylation of the protein upon contact of the sample with urea. In Figure 3D an

Figure 6 Partial 2D gel images showing the enrichment of enolase by hydrophobic interaction chromatography. (A) Total extract; (B) proteins from a fraction collected from the column.

Figure 7 Partial 2D gel images showing the enrichment of low abundance proteins of H. influenzae by heparin chromatography. (A) Total extract; (B, C) proteins from fractions collected from the heparin column. The arrowheads indicate spots representing two proteins (B, topoisomerase I; C, ATP-dependent RNA helicase) which are not visible in total extract (A).

Figure 7 Partial 2D gel images showing the enrichment of low abundance proteins of H. influenzae by heparin chromatography. (A) Total extract; (B, C) proteins from fractions collected from the heparin column. The arrowheads indicate spots representing two proteins (B, topoisomerase I; C, ATP-dependent RNA helicase) which are not visible in total extract (A).

example of protein heterogeneity, most likely due to glycosylation, is presented.

Following 2D electrophoresis, proteins can be identified by mass spectrometric analysis of the peptides resulting from the in-gel digestion with a specific protease, such as trypsin. In another approach, the proteins can be electrotransferred onto membranes and identified by immunoreaction with specific antibodies, by N-terminal sequencing or amino acid composition analysis. For those proteins for which the

Table 2 Steps in the preparation of 2D electrophoresis

IPG strip rehydration and sample preparation

Protein extraction, centrifugation, recovery in sample solution

Sample application

Application in cups at either or at both ends of the strip or strip rehydration in a solution containing the protein sample

First dimensional separation (isoelectric focusing)

Start at 200 V and increase gradually to 5000 V; keep 5000 V for 6-48 h, depending on sample, quantity and strip range; narrow pH range strips require longer focusing times

Reduction and alkylation of proteins on IPG strip

Equilibration of strip with reducing and alkylating agents or freeze until use

Second dimensional separation (SDS-PAGE)

Preparation of gel of the desired acrylamide concentration; gels should carry a label to identify them afterwards; establishment of contact between strip and gel with agarose solution; run at 40 mA/gel

Protein fixing and staining or blotting

Fixation of proteins within the gel and staining with silver or Coomassie blue or drying of the gel and exposure to a film or phosphorimager for detection of radiolabelled proteins or electrotransfer of proteins to membranes for immunoblot, MS or amino acid analysis

Gel scanning

Storage of image in a database

Gel comparison

Gel comparison and protein quantification using specific software; comparison with database master gels via the WorldWideWeb Identification of proteins

Identification of protein spots from gels by mass spectrometry or from membranes by N-terminal sequencing, amino acid composition analysis, MS or immunoblots genomic sequence is in a database, the most efficient identification method presently available is matrix-associated laser desorption ionization mass spectrometry (MALDI-MS) with which about 500 spots can be analysed daily by one person. The method tolerates small amounts of salt in the sample, so that no time-consuming desalting steps are required after digestion. Several approaches using a combination of protein digestion on membranes and MS have also been reported. Table 2 summarizes the essential steps of 2D electrophoresis and protein analysis.

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