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Lee Lomas

Publications and source records attributed to Lee Lomas.

10 recordsLinked to original sources
PubMed2006

Reducing protein concentration range of biological samples using solid-phase ligand libraries.

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.

Chromatography, Ion Exchange↗
PubMed2006

Protein Equalizer Technology : the quest for a "democratic proteome".

No proteome can be considered "democratic", but rather "oligarchic", since a few proteins dominate the landscape and often obliterate the signal of the rare ones. This is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is seen again and again. A host of pre-fractionation techniques have been described, but all of them, one way or another, are besieged by problems, in that they are based on a "depletion principle", i.e. getting rid of the unwanted species. Yet "democracy" calls not for killing the enemy, but for giving "equal rights" to all people. One way to achieve that would be the use of "Protein Equalizer Technology" for reducing protein concentration differences. This comprises a diverse library of combinatorial ligands coupled to spherical porous beads. When these beads come into contact with complex proteomes (e.g. human urine and serum, egg white, and any cell lysate, for that matter) of widely differing protein composition and relative abundances, they are able to "equalize" the protein population, by sharply reducing the concentration of the most abundant components, while simultaneously enhancing the concentration of the most dilute species. It is felt that this novel method could offer a strong step forward in bringing the "unseen proteome" (due to either low abundance and/or presence of interference) within the detection capabilities of current proteomics detection methods. Examples are given of equalization of human urine and serum samples, resulting in the discovery of a host of proteins never reported before. Additionally, these beads can be used to remove host cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with "equalizer beads", these last impurities can be effectively removed at a small cost and with minute losses of the main, valuable product.

Biomarkers↗
PubMed2005

Proteome analysis in the clinical chemistry laboratory: myth or reality?

BACKGROUND: We review here modern aspects of proteomic analysis, as displayed via orthogonal mass/charge analysis (isoelectric focusing in the first dimension, followed by sodium dodecyl sulphate electrophoresis in polyacrylamide gels, SDS-PAGE, at right angles, in the second dimension). METHODS: This technique is capable of displaying a few thousand polypeptide chains, characterized by a single pI and M(r) value as coordinates, and recognized via elution, digestion and mass spectrometry analysis. Although, up to the present, this technique has been used mostly for advanced research, with no immediate applications in the clinical chemistry laboratory, there are hints that such applications will soon become a reality. RESULTS AND CONCLUSIONS: In the field of cancer research, it is here shown that stathmin (Op18) becomes heavily phosphorylated in cancerous mantle cell lymphomas and that the progression of the disease can be followed by the progression of phosphorylation of Op18 and by the appearance of additional phosphorylated spots. Also chemoresistance of different tumors has been evaluated via 2D-PAGE through quantitative, differential proteomics: among up- and down-regulated proteins in a human cervix squamous cell carcinoma cell line (A431), rendered resistant to cisplatin, one particular protein was found to appear in large quantities by de novo synthesis: 14-3-3, a protein known to impart resistance to apoptosis to cells. In the field of brain disorders, we could set up an easy test for detecting pathological prions in sporadic Creutzfeldt-Jakob disease (sCJD), by simply searching for those pathological forms in the olfactory mucosa (up to this finding, diagnosis could only be confirmed post-mortem). We are currently working on a test for differentiating sCJD from all the other degenerative dementias. Upon 2D mapping of cerebrospinal fluid (CSF) and immunoblot analysis, we could identify a major spot (pI 4.8, M(r) 30 kDa) followed by some two-three minor spots (pIs 5.0-6.0, same M(r) value) of the same 14-3-3 anti-apoptotic protein involved in chemoresistance. By this test, sCJD could be differentiated from all the other degenerative dementias, which are 14-3-3 negative (in sCJD, the rapid and massive brain cell damage releases large quantities of 14-3-3 in the cerebrospinal fluid). Another protein that appears very promising as a marker for sCJD is cystatin C, that is strongly up-regulated in this pathology. Human sera should also be mined for discovery of many more markers for disease. Up to the present, no one could be found, but this was due to the presence of several major proteins, obscuring all rare ones. Via several immuno-subtraction steps, followed by ion exchange and size exclusion chromatography, one can now detect proteins and peptides present in sera at levels below 10 ng/mL, highlighting the road to discovery of novel markers of disease. Another technique that could revolutionize biomarker discovery in biological fluids consists in the use of combinatorial beads to reduce the dynamic range. They consist in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of amino acids, resulting in millions different structures. When these beads are impregnated with complex proteomes (e.g., human sera, CSF, urines) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility of evidencing low-abundance species.

Animals↗
PubMed2005

Classification of cancer types by measuring variants of host response proteins using SELDI serum assays.

Protein expression profiling has been increasingly used to discover and characterize biomarkers that can be used for diagnostic, prognostic or therapeutic purposes. Most proteomic studies published to date have identified relatively abundant host response proteins as candidate biomarkers, which are often dismissed because of an apparent lack of specificity. We demonstrate that 2 host response proteins previously identified as candidate markers for early stage ovarian cancer, transthyretin and inter-alpha trypsin inhibitor heavy chain 4 (ITIH4), are posttranslationally modified. These modifications include proteolytic truncation, cysteinylation and glutathionylation. Assays using Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS) may provide a means to confer specificity to these proteins because of their ability to detect and quantitate multiple posttranslationally modified forms of these proteins in a single assay. Quantitative measurements of these modifications using chromatographic and antibody-based ProteinChip array assays reveal that these posttranslational modifications occur to different extents in different cancers and that multivariate analysis permits the derivation of algorithms to improve the classification of these cancers. We have termed this process host response protein amplification cascade (HRPAC), since the process of synthesis, posttranslational modification and metabolism of host response proteins amplifies the signal of potentially low-abundant biologically active disease markers such as enzymes.

Algorithms↗
PubMed2005

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

Adsorption↗
PubMed2004

Simultaneous monitoring of multiple kinase activities by SELDI-TOF mass spectrometry.

Cellular response to the external environment is often controlled by one or more protein kinases. We report a methodology for simultaneously monitoring multiple kinase activities across multiple signal-transduction pathways using ProteinChip Array technology. Based on the addition of specific peptide reporters, kinase activity is detected by the presence of a mass shift of 80 Da (or multiple thereof) corresponding to the addition of one or more phosphate groups. These phosphorylated peptide substrates are then enriched using an immobilized metal affinity capture (IMAC)-Ga array and detected directly by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). SELDI-TOF MS is sensitive, tagless (nonradioactive, nonfluorescent), can be easily multiplexed for the analysis of several different kinases in a single reaction mixture (limited only by the specificity of the kinase for its substrate peptides), and is directly scalable through the use of robotic sample processing. By multiplexing kinase assays, one can dramatically increase the amount of information obtained from rare or volume-limited samples. More important, results reflect closely the complex interrelationships between kinases and show high correlation with in vivo assays.

Enzyme Inhibitors↗
PubMed2002

Application of surface-enhanced laser desorption/ionization technology to the detection and identification of urinary parvalbumin-alpha: a biomarker of compound-induced skeletal muscle toxicity in the rat.

In toxicity studies, compound-induced changes are typically evaluated using a combination of endpoints and there are often a number of potential markers in biological fluids which can indicate toxic change in tissues and organs. However, some biomarkers are not specific to the organ of injury and therefore there is a continuing search for more sensitive and specific indicators of target organ toxicity. In experiments to assess the potential diagnostic usefulness of surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, skeletal muscle toxicity was induced in Wistar Han rats by administering 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD). The skeletal muscle toxicity was monitored using established endpoints such as increase in serum aldolase (Aldol), aspartate aminotransferase (AST) and histopathology, and also using SELDI retentate chromatography mass spectrometry of urine samples. Clear differences in urinary protein patterns between control and TMPD-treated animals were observed on the ProteinChip surfaces. Additionally a specific urine marker protein of 11.8 kDa was identified in TMPD-dosed rats, and the detection of the marker was related to the degree of skeletal muscle toxicity assessed by recognized clinical pathology endpoints. The 11.8 kDa protein was identified as parvalbumin-alpha. These experiments demonstrated the potential of urinary parvalbumin-alpha as a specific, noninvasive, and easily detectable biomarker for skeletal muscle toxicity in the rat and the potential of SELDI technology for biomarker detection and identification in toxicology studies.

Animals↗
PubMed2002

SELDI ProteinChip array in oncoproteomic research.

Research into the causes, early detection and treatment of cancers is a primary focus of the health care industry and proteomic-based methodologies are providing an increasingly important role in addressing these issues. The ProteinChip Array technology forms the basis of a clinical proteomics platform designed to expedite the discovery, validation, and characterization of cancer biomarkers at all stages of cancer progression. Being able to detect cancer progression early in turn allows for the possibility of more effective treatment. This short review serves to introduce the technology by highlighting specific examples related to cancer biomarker discoveries.

Biomarkers, Tumor↗
PubMedDate not supplied

Exploring the hidden human urinary proteome via ligand library beads.

The human urinary proteome has been reassessed and re-evaluated via a novel concentration/equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species.

Adult↗