Peptides quantitative analysis

Ji, J. Chakraborty A. Geng, M. Zhang, X. Amini, A. Bina, M. Regnier, F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 2000, 745,197-210. 

Currently, LC-MS is widely used for the analysis of polar compounds, such as medicinal metabolites and bioactive peptides, since the interface has been improved and several new ionization methods have been developed. The sensitivity and reproducibility are sufficient for a daily quantitative analysis. The usefulness of the LC-MS has been demonstrated for studies on Type II pheromones using a time-of-flight MS with electrospray ionization (ESI) [180]. Each epoxydiene derived from the (Z3,Z6,Z9)-triene shows three ion series of [M+NHJ+, [M+H]+, and [M-OH]+ with high resolution and good sensitivity, indicating its molecular formula. In addition to these, characteristic fragment... 

For MK2, one may use a peptide substrate (see, e.g., Manke et al, 2005) or recombinant heat shock protein (hsp) 25 (Stokoe et al, 1992). For the peptide substrate, analysis is as described for the p90RSK assay. If using hsp27 as substrate, samples of the reaction products are analyzed by SDS-PAGE and autoradiography or quantitation performed by phosphorimager. 

J Gerhardt, K Nokihara, R Yamamoto. Design and applications of a novel amino acid analyzer for D/L and quantitative analysis with gas chromatography, in JA Smith, JE Rivier, eds. Peptides Chemistry and Biology. Proceedings of the 12th American Peptide Symposium, Escom, Leiden, 1992, pp 457-458. 

NMR is a remarkably flexible technique that can be effectively used to address many analytical issues in the development of biopharmaceutical products. Although it is already more than 50 years old, NMR is still underutilized in the biopharmaceutical industry for solving process-related analytical problems. In this chapter, we have described many simple and useful NMR applications for biopharmaceutical process development and validation. In particular, quantitative NMR analysis is perhaps the most important application. It is suitable for quantitating small organic molecules with a detection limit of 1 to 10 p.g/ml. In general, only simple one-dimensional NMR experiments are required for quantitative analysis. The other important application of NMR in biopharmaceutical development is the structural characterization of molecules that are product related (e.g., carbohydrates and peptide fragments) or process related (e.g., impurities and buffer components). However, structural studies typically require sophisticated multidimensional NMR experiments. 

Marquez CD, Weintraub ST, Smith PC. 1997. Quantitative analysis of two opiod peptides in plasma by liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr Biomed Sci Appl 694 21. 

Gusev, A. I. et al.. Direct quantitative analysis of peptides using matrix assisted laser desorption ionization. Anal. Bioanal. Chem., 354, 455, 1996. 

Raman spectroscopy is a related vibrational spectroscopic method. It has a different mechanism and therefore can provide complementary information to infrared absorption for the peptide protein conformational structure determination and some multicomponent qualitative and/or quantitative analysis (Alix et al. 1985). 

How often an LC-MS should be calibrated depends on the mass accuracy required. For example, instrument calibration should be verified daily when performing accurate mass measurements of peptides and proteins. However, the quantitative analysis of small molecules requires less frequent calibration. 

Extraction is an essential step when analyzing solid samples. In some cases homogenization with a solvent suffices, but in others the sample must first be coimninuted. Water, solutions of acetic acid or sodium chloride, or more complex saline solutions are used as solvents. Mixtures of water and methanol or water and ethanol are also employed. The choice of solvent depends on the degree of selectivity desired in the extraction and whether the extraction yield is intended for quantitative analysis. Optimization of the extraction procedure is required in all cases, to fit the nature of the sample to be analyzed and the range of molecular weights of the peptides to be separated. For example, water has been used as the extraction solvent for cheese (33) and legumes (34). Saline solutions have been utilized to extract peptides from meat (35-38) and flour (39,40). Benedito de Barber et al. (41) examined differences in the extractability of amino acids and short peptides in various solvents (1M acetic acid, 70% ethanol, and distilled water) they concluded that extraction with 1M acetic acid yielded the maximum amino acid and peptide contents. 

Gel-free approaches employ liquid chromatography followed by mass spectrometry to analyze peptides in order to assess protein levels in complex samples such as those derived from microbial extracts or environmental samples. This approach allows for quantitative analysis of identified proteins,... 

Amphipathic peptides may stick to amphipathic surfaces, i.e., cells, media components, or chemical instruments, thus making their quantitative analysis rather difficult (35). 

The major limitation of both UV and MS detectors is that neither can provide quantitative or even semiquantitative information without reference standards. Ultraviolet response depends on the presence of a chromophore in a molecule and evidently might vary from one molecular species to another in a library. Although successful application of electrospray mass spectrometry for quantitative analysis of peptides has been reported [35], one should always keep in mind that signal intensity in a mass spectrum depends on the ability of a molecule to ionize. The ability to produce ions, especially with soft ionization techniques, might be very different for different molecules within one library, and the difference might be even bigger from one library to another. 

Radioimmunoassay (or RIA) was first introduced as an analytical technique in 1959. Since then it has played an increasingly important role in the quantitative analysis of hormones and drugs. Much of the recent rapid progress in endocrinology may in fact be attributed to the availability of this sensitive and accurate method for quantitation of steroidal and pro-tein/peptide hormones. 

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