Biomedical subjects

Ben Herbert

Publications and source records attributed to Ben Herbert.

7 recordsLinked to original sources

PubMed2003

Fungal proteomics: mapping the mitochondrial proteins of a Trichoderma harzianum strain applied for biological control.

Mitochondria are essential for cellular functions across organisms. A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties. We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination. We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T. harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry. Over 50% of the identified proteins were known to localize in the mitochondria. Three protein spots were identified from T. harzianum and a further five protein spots from the genera Trichoderma and Hypocrea. The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In total, we identified 25 protein spots from T. harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases. To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus.

Chromatography, Liquid↗

PubMed2003

Soft immobilized pH gradient gels in proteome analysis: a follow-up.

As a follow-up of a previous work on two-dimensional map analysis utilizing soft (< 4%T) immobilized pH gradient (IPG) matrices in the first dimension (Candiano et al., Electrophoresis 2002, 23, 292-297), we have further optimized the preparation of such dilute IPG gels. One important step for obtaining an even reswelling of the entire IPG strip along the pH 3-10 interval is a washing step in 100 mM citric acid. It appears as though after rinsing off the excess acid in distilled water, a gradient of this tricarboxylic acid remains trapped into the IPG matrix, from almost nil at the acidic gel region to substantially higher amounts in its basic counterpart. This gradient helps in obtaining a uniform reswelling of the IPG strip, since carboxyl groups are more heavily hydrated than amino groups. The combined effects of uniform reswelling and of diluting the gel matrix favor penetration of large macromolecules (> 200 kDa) and allow for better spot resolution and for the display of a substantially higher number of spots also in the 30-60 000 Da region. A delipidation step in tri-n-butylphosphate:acetone:methanol (1:12:1) appears to substantially improve spot focusing and greatly diminish streaking and smearing of spots in all regions of the pH gradient.

Blood Platelets↗

PubMed2003

Beta-elimination: an unexpected artefact in proteome analysis.

Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.

Alkylation↗

PubMed2003

Prefractionation techniques in proteome analysis.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Chemical Fractionation↗

PubMed2002

Two-dimensional maps in soft immobilized pH gradient gels: a new approach to the proteome of the Third Millennium.

Same major improvements in proteome analysis of cytosolic and membrane proteins by two-dimensional mapping are here reported. A much improved transfer of proteins from the first to the second dimensional sodium dodecyl sulfate (SDS)-gel is obtained by simply diluting the gel matrix, normally composed of 4%T polyacrylamide in all commercially available Immobiline strips down to as low as 3%T. In the analysis of total lysates of platelets, this augmented transfer has been evaluated as being 2-3 times higher than in standard 4%T gels. A second major improvement, in the case of analysis of membrane protein preparations, has been demonstrated to consist in a delipidation step in a tertiary solvent mixture composed of tri-n-butyl phosphate:acetone:methanol in a 1:12:1 ratio. By adopting this protocol, large amounts of spectrins (240-220 kDa, filamentous proteins of the red blood cell membranes) could be transferred vs. essentially none when delipidation was omitted. The present report also confirms the importance of a reduction and alkylation step of the protein sample prior to all electrophoretic steps, including focusing in the Immobiline gel, as recently reported by Herbert et al.

Acrylic Resins↗

PubMed2002

Proteomics and immunomapping of reactive lymph-node and lymphoma.

In the present study we show that two-dimensional (2-D) maps together with immuno-detection allow the precise identification of important leukocyte differentiation and tumor markers (e.g., CD3 and CD5), and important cell cycle regulatory molecules such as cyclin dependent kinases, notably CDK6. In addition, the comparative evaluation of molecular expression (e.g., CD5) in maps developed with normal and lymphoma samples can provide reproducible and precise information regarding the molecular expression in different cell populations. Accordingly, we could detect a much increased level of expression of CD5 in mantle cell lymphoma, up to ten times higher than in the control. In addition, CD5 in tumor tissues seems to be microheterogeneous as compared to normal samples.

Electrophoresis, Gel, Two-Dimensional↗

PubMedDate not supplied

Carbamylation of proteins in 2-D electrophoresis--myth or reality?

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

Alkylation↗