Protein bioanalysis

Various methods were evaluated for the targeted proteomics of human growth hormone (hGH) in human plasma [111]. hGH was spiked in plasma 10-fold above natural level ( 16 pg/pl). Iiutially, the full plasma proteome was reduced, alkylated, and digested prior to LC-MS via DDA on an ion-trap instrument. hGH could be identified from its T, peptide. Next, the plasma proteome was fractionated by RPLC and GE prior to digestion and LC-MS analysis. hGH could be identified with higher confidence. Finally, an LCxLC-MS approach was apphed, which enabled hGH identification from five tryptic peptides. An important conclusion was that hGH could be detected in a complex sample at the low femtomole level among proteins that were 40,000 x more abundant. The results show that a multidimensional approach may be taken for targeted proteomics and quantitative protein bioanalysis. 

Protein bioanalysis Granulocyte colony-stimulating factor (GCSF) Plasma Fluorescence-labeled antibodies, affinity chromatography 0.6 nmol/L 10... 

In summary, despite the significant challenges associated with LC-MS/MS-based assays for protein drug quantitation, these assays stiU show promise as an emerging complementary technique for protein bioanalysis. 

Ruan, Q., Ji, Q.C., Arnold, M E., Humphreys, W.G., Zhu, M. (2011) Strategy and its implications of protein bioanalysis utilizing high-resolution mass spectrometric... 

Algar WR, Krull UJ (2008) Quantum dots as donors in fluorescence resonance energy transfer for the bioanalysis of nucleic acids, proteins, and other biological molecules. Anal Bioanal Chem 391 1609-1618... 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

Widely used in bioanalysis for measuring small amounts of drug and for studying drug-protein binding. 

Capillary electrophoresis has become very successful in the bioanalysis of proteins but researchers are often looking for ways to improve the various techniques,... 

It is well known that a great variety of biomolecules exist where metals and metalloids are bound to proteins and peptides, coordinated by nucleic acids or complexed by polysaccharides and small organic ligands such as organic acids.55 Most proteins contain amino acids with covalently bonded heteroelements such as sulphur, selenium, phosphorus or iodine.51 Several reviews have been published on the development of mass spectrometric techniques for bioanalysis in metal-lomics , which integrate work on metalloproteins, metalloenzymes and other metal containing biomolecules.1 51 53 54 56-59 The authors consider trace metals, metalloids, P and S (so-called... 

C. Albrecht, R. Reichen, J. Visser, D. K. F. Meijer, and W. Thormann, Differentiation between naproxen, naproxen-protein-conjugates and naproxen-lysine in plasma via micellar electrokinetic capillary chromatography—A new approach in the bioanalysis of drug targeting preparations, Clin. Chem. 43 2083-2090 (1997). 

Fig. 5 Reversible oxidation and reduction of electroactive SAMs to generate renewable microarray surfaces for bioanalysis left). Corresponding fluorescent micrographs (right) depicting three cycles of carbohydrate and protein immobilization and release on the same substrate...

The analytes of interest in quantitative bioanalysis vary, from small molecules with molecular weights usually less than 1,000 Da (e.g., drugs and their metabolites) to large biopolymers, such as proteins. The focus of this chapter is on the quantitation of small molecules in biological samples by LC-MS, though some of the principles presented in this chapter are also applicable to the quantitative analysis of large molecules in biological samples. 

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