Analysis and quantification of biomolecules

Understanding Bio analytical Chemistry Principles and applications Victor A. Gault and Neville H. McClenaghan 2009 John Wiley Sons, Ltd 

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High-performance liquid chromatography (HPLC) has emerged as one of the most useful tools for the bioanalytical laboratory. This is a powerful stand-alone method that can be used for the purification, separation and quantification of biomolecules. Notably HPLC analysis can be combined with other high-tech methods such as mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy to enable the identification of biomolecules. 

While straight MS analysis can yield important information in terms of identification, characterization and quantification of biomolecules, it becomes a much more powerful tool with further MS or when combined with other separation technologies. As noted earlier, these approaches include MS/MS, GC-MS, LC-MS and CE-MS. These methods have been extensively exploited in virtually all aspects of bioanalysis, and while fundamentally useful for peptide and protein analysis, these methods have also been used in the analysis of lipids, nucleic acids and a wide range of small molecules and drugs. The range of applications is obviously outside the scope of a book like this, but some indications of the uses of each of these techniques are given below. 

Lisboa et al. extensively exploited the possibilities of XPS analysis to study different graft polymer layers prepared by UV-induced free radical polymerization on poly (pyrrole) (PPy) films [9]. The aim was to prepare carboxylic acid or amine functionalized PPy for biomolecule immobilization. The contribution of the PPy carbon atoms in the Cls peak disappeared after a grafting time of 1200 s for acrylic acid and almost completely disappeared after 1500 s for allylamine, indicating that the PPy surface was completely covered with the graft polymer. For quantification of the amount of carboxylic acid and amine functionalities, a derivatization of the functional groups with trifluoroetha-nol and 4-trifluoromethylbenzaldehyde, respectively, was necessary. 

In both tandem-in-space and tandem-in-time instruments, the most common experiment is for the first analyzer to select specific ions from the total ion beam arriving from the ion source. Next, the selected ions undergo collision-induced dissociation (CID) in a pressurized cell followed by the analysis of the product ions in the second analyzer. In tandem-in-time the same analyzer is used for both scans, but at different times. The resulting product ion spectra (or precursor and neutral loss spectra in other forms of MS/MS analysis) provide vital structural information for the identification of small molecules (such as drug metabolites) as well as complex biomolecules. Selected reaction monitoring (SRM), another mode of MS/MS operation, provides highly specific and sensitive quantification of target analytes. 

High-performance liquid chromatography-mass spectrometry (HPLC-MS) is a powerful analytical technique widely used in recent years for the analysis of biomarkers and metabolites. Biomarker determination and quantification, whether metabolic or adducted biomolecules, are commonly used to evaluate exposure and support biomonitoring research, especially in the area of occupational exposure and health. Some of the common problems and strategies of HPLC-MS biomarker analysis involve matrix effects, the use of isotope-labeled internal standard compounds, and sample cleanup usually all of these factors must be evaluated within the development phase of an analysis procedure. Specific examples of biomarker analysis using HPLC-MS include acrylamide, aromatic compounds, and 1-bromopropane, and these examples are discussed in detail. 

Mass spectrometry (MS) in combination with the chromatographic separation techniques of gas chromatography (GC) or liquid chromatography (LC) have become the gold standard methods for the analysis of steroids [1-3], These methods allow the identification of novel steroids and in combination with reference standards quantification. Biological MS stretches back to the 1950s, when steroids was one of the first class of biomolecules analyzed by MS [4-9],... 

If some of the physico-chemical properties of nucleic acids pose many challenges to their analysis by MALDI-TOF, then some others afford intrinsic advantages as compared to proteins /peptides and other biomolecules. For instance, their primary structure is much more homogeneous than that of proteins, consisting of only four relatively similar building blocks. Because of this structural simplicity and homogeneity, relative and even absolute (with an internal standard) quantification can be readily accomplished and this constitutes the basis for a number of assays (as discussed below). The remainder of this chapter is mostly devoted to descriptions of the different assays that have been developed for the analysis of NAs, under the somewhat restrictive boundary conditions of UV-MALDI-TOF-MS. 

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