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 Mr 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, Mr 30 kDa) followed by some two–three minor spots (pIs 5.0–6.0, same Mr 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.

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

RIGHETTI, Piergiorgio;CASTAGNA, Annalisa;ANTONUCCI, Francesca;PIUBELLI, Chiara;CECCONI, Daniela;CAMPOSTRINI, Natascia;RUSTICHELLI, Chiara;ANTONIOLI, Paolo;ZANUSSO, Gianluigi;MONACO, Salvatore;
2005-01-01

Abstract

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 Mr 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, Mr 30 kDa) followed by some two–three minor spots (pIs 5.0–6.0, same Mr 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.
Proteome; clinical chemistry; proteomics; Proteome analysis; Stathmin; Mass spectrometry analysis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/301236
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