Ben Herbert

Advances in protein solubilisation for two-dimensional electrophoresis

Two‐dimensional (2‐D) electrophoresis remains the highest resolution technique for protein separation and is the method of choice when complex samples need to be arrayed for characterisation, as in proteomics. However, in current proteome projects the total number of proteins identified from 2‐D gels is often only a small percentage of the predicted proteome. In addition, there is an almost complete lack of hydrophobic proteins on 2‐D gels, especially those using immobilised pH gradients. Recently there have been a number of publications reporting reagents which improve protein solubilisation prior to isoelectric focusing. The improved solubilization possible with these reagents has increased the total number of proteins able to be visualised on 2‐D gels and also allowed the separation of hydrophobic proteins, such as integral membrane proteins.

Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how?

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF)‐mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to “scrambled” disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human α‐ and β‐globin chains, which possess only one (α‐) or two (β‐) ‐SH groups. As a result, misplaced spots are to be found in the resulting two‐dimensional (2‐D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their pI, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS‐PAGE step.

Proteomics: Capacity versus utility

Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High‐throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.

Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.

Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two‐dimensional (2‐D) gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2‐D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2‐D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2‐D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.

Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica Expansion of a Repertoire of Virulence-associated Factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser↓His motif for autocatalytic cleavage by cathepsin Ls were preserved.

What place for polyacrylamide in proteomics

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.

All existing protocols for protein separation by two‐dimensional (2‐D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) to monitor protein alkylation by iodoacetamide over the period 0 – 24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached ∼70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2‐D gel analysis in particular and proteomics in general are discussed.

Proteomic response of the biological control fungus Trichoderma atroviride to growth on the cell walls of Rhizoctonia solani

Trichoderma atroviride has a natural ability to parasitise phytopathogenic fungi such as Rhizoctonia solani and Botrytis cinerea, therefore providing an environmentally sound alternative to chemical fungicides in the management of these pathogens. Two-dimensional electrophoresis was used to display cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls. Protein profiles were compared to identify T. atroviride proteins up-regulated in the presence of the R. solani cell walls. Twenty-four protein spots were identified using matrix-assisted laser desorption ionisation mass spectrometry, liquid chromatography mass spectrometry and N-terminal sequencing. Identified up-regulated proteins include known fungal cell wall-degrading enzymes such as N-acetyl-β-d-glucosaminidase and 42-kDa endochitinase. Three novel proteases of T. atroviride were identified, containing sequence similarity to vacuolar serine protease, vacuolar protease A and a trypsin-like protease from known fungal proteins. Eukaryotic initiation factor 4a, superoxide dismutase and a hypothetical protein from Neurospora crassa were also up-regulated as a response to R. solani cell walls. Several cell wall-degrading enzymes were identified from the T. atroviride culture supernatant, providing further evidence that a cellular response indicative of biological control had occurred.

Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.

Although it is highly recommended that reduction and alkylation of free –SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA).The present matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free –SH group of Cys in proteins at alkaline pH values (pH 8.5–9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the –SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.

Two-Dimensional Electrophoresis: The State of the Art and Future Directions

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is the only method currently available which is capable of simultaneously separating thousands of proteins. Thus 2-D PAGE is the heart of proteome technology. The first dimension of 2-D PAGE is isoelectric focusing (IEF), during which proteins are separated in a pH gradient until they reach a stationary position where their net charge is zero. The pH at which a protein has zero net charge is called its isoelectric point (pI). In the second dimension the proteins separated by IEF are separated orthogonally by electrophoresis in the presence of sodium dodecyl sulphate (SDSPAGE). The surfactant SDS binds to proteins, overriding their intrinsic charge, such that they all have the same charge density and free solution electrophoretic mobility. When the SDS-coated proteins migrate in a sieving polyacrylamide gel they separate based on their molecular mass. The high resolution of 2-D PAGE results from the first and second dimension separations being based on independent protein parameters.