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A Quantitative Method for the Determination of Proteins in Complex Bioanalysis studies

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A quantitative method for the determination of proteins in complex bioanalysis studies has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma.

This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography–mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 ?l of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.

Protein phosphorylation catalyzed by protein kinase is one of the most important post-translational modifications and plays a significant regulatory role in many vital bioanalysis studies. Aberrant protein-phosphorylation states and kinase activity are closely associated with many human diseases. Monitoring the kinase activity and its inhibition is essential for fundamental biochemical research and kinase-targeted drug discovery. Nanomaterial-based kinase assays provide a promising toolkit for exploring protein kinase functions, which have attracted growing interest in academic research, biomedical diagnosis, and pharmaceutical discovery. The recent advances in the development of the protein kinase activity assays based on various nanomaterials, classifies these methods by different analytical techniques, summarizes the general design strategies, and offers perspectives on future developments.

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