Isotope labelling advances

An important advance on these studies was the possibility of isolating AORs from Fe enriched media with obvious interest for an iron-sulfur center site labeling, with enhanced sensitivity of the Mossbauer studies. The work developed with bacterial systems is advantageous as compared with mammalian systems for isotopic labeling and opens the possibility of a direct measurement of substrate binding. Spectra of the enzyme in oxidized, partially reduced, benzaldehyde-reacted, and fully reduced states were recorded at different temperatures and with variable externally applied magnetic fields (222). In the oxidized enzyme, the clusters are diamag-... 

Figure 16.4 A more advanced ICAT design uses an acid-cleavable spacer arm to facilitate elution of labeled peptides from a (strept)avidin affinity column. The use of 14C isotopes instead of deuterium labels permits precise reverse phase separations prior to mass spec that show no elution peak time differences between isotope-labeled and normal atom-labeled peptides.

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

Hunger and Wang provide an account of advances in the characterization of solid catalysts in the functioning state by nuclear magnetic resonance spectroscopy. Examples include investigations of zeolite-catalyzed reactions with isotopic labels that allow characterization of transition states and reaction pathways as well as characterization of organic deposits, surface complexes, and reaction intermediates formed in catalyst pores. 

It is curious that there have been more fundamental developments in 1,3,5-triazine synthesis by this method than in any of the other sections, yet it is the oldest of all the synthetic routes. Cyanuric acid was originally prepared by the pyrolysis of uric acid (7) (see Section 2.20.1.2). The mechanism was proposed by Brandenberger and coworkers on the basis of isotopic labelling studies, and was reviewed by Modest (B-61MI22000, p. 706). The rearrangement of pyrimidines to 1,3,5-triazines is the most important advance in this area. 

Several biosynthetic proposals have been advanced, based on patterns of occurrence, known in vitro reactivity of bisbenzylisoquinoline alkaloids, and mechanistic analogy. Although unconfirmed (e.g., by isotopic labeling), these theories point the way toward further research. 

Even with advanced methods, several models may satisfy the Qm n requirement. Further discrimination necessitates additional information. This may come from experience, analogy, measurements using special techniques, such as isotope labeling, and fundamental knowledge on chemical mechanisms. 

Quantitative analysis has become possible due to technical advances in synthesis of complex molecules with isotopic labels at any one of many specific position and measurements of KIE determined accurately and precisely by mass-spectrometry and radioactive methods. The most informative method for elucidation of the enzyme reaction limiting step and nature of transition-state is the competitive labeled method (Schramm, 1999). This method is based on the use of two labeled preparations of the same substrate, one with the labeled atom at a site expected to experience bonding changes at the TS and a second preparation with a different labeled atom at a site remote from the bond-breaking site. Many molecules of interest can be specifically labeled with radioactive atoms T or I4C and can be incorporated into substrates that also contain stable isotopes D, 15N and 180. 

In quantitative proteomics, two alternative strategies have been developed. The first one is based on 2-D PAGE combined with mass spectrometry for protein identification (Haynes and Yates, 2000). This method and current advances in the differential display of proteins by 2-D PAGE are discussed at length in another chapter of this book. The second approach exploits mass spectrometry in combination with stable isotope labeling for gaining accurate quantitative information on proteins. Quantitative MS via stable isotope labeling of proteins... 

Methoxypyrazines are compounds with very low detection thresholds which must be determined at very low levels. For these compounds, different selective isolation methods have been proposed (Allen et al. 1994 Sala et al. 2002). Some authors use a simple extraction (Kotseridis et al. 1999 Falcao et al. 2007) or an optimized headspace SPME procedure (Chapman et al. 2004 Prouteau et al. 2004) using in most cases isotopically-labelled internal standards to compensate for matrix effects. In spite of the claims of the authors, all these methods present some difficulties to accurately determine the compounds at the lowest levels at which they can be found. A recent report has presented an advanced method combining the preconcentration ability of headspace SPME with the selectivity of comprehensive GC (Ryan et al. 2005). 

Advances in NMR spectrometer and probe technology and in solid-state NMR methods, along with development of new pulse sequences have opened up the biomolecular NMR field to the study of membrane-bound proteins and large molecular weight (MW > 50 kDa) systems. In addition, studies of low-sensitivity nuclei are expected to gain in popularity as the appropriate technical and experimental expertise is developed and refined. Another important area concerns developments in protein engineering that allow preparation of biomolecules isotopically labeled either uniformly or at particular sites. The rapid and continuing development of these... 

Feeding experiments with isotopically labeled precursors have shown that many NR fungal polyketides are formed by the use of advanced starter units. In the classic case of norsolorinic acid 7 biosynthesis, it has long been known that hexanoate forms the starter unit. Differential specific incorporation of acetate into the early and late positions in compounds such as citrinin 3 have been used to argue that these compounds may have been formed by more than one PKS so that one PKS makes an advanced starter unit, which is passed to a second PKS for additional extension. 

Another method to introduce unnatural amino acids into a polypeptide chain is through complete chemical synthesis (10). The predominantly used method, stepwise solid-phase peptide synthesis (SPPS), attaches the C-terminal amino acid to a solid support, and amino acids are added one at a time to the N-terminus. A clear advantage of chemical synthesis is that it enables the accurate introduction of unnatural amino acids at any site in a protein. The number of unnatural amino acids that can be introduced is limited only to the size of the chain, and chains of entirely unnatural amino acids can be produced using this method. Chemical synthesis is useful particularly for the incorporation of isotopic labels and unnatural amino acids that are toxic to cells or incompatible with the translational machinery. However, construction of a polypeptide chain using even the most advanced chemical synthesis techniques is daunting when confronted with the construction of an entire protein, as these methods currently are limited to approximately 100 amino acids (10). 

Kinetic steps are best identified by measuring the initial products formed from individual species (including postulated intermediates) or from simple mixtures. Isotopically labeled species have proved useful in such experiments. Initial products of homogeneous processes are observable in batch reactors at sufficiently short times or in flow reactors at points sufficiently near the inlet. The most advanced systems for initial product detection are molecular beam reactors (Herschbach 1976 Levine and Bernstein 1987) in which specific collisions are observed. Each of these techniques restricts the number of contributing reactions in a given experiment, so that their stoichiometry and rates can often be inferred. 

---