Isotope-labeled amino acids

Veenstra, T. D. Martinovic, S. Anderson, G. A. Pasa-Tolic, L. Smith, R. D. Pro-teome analysis using selective incorporation of isotopically labeled amino acids. /. Am. Soc. Mass Spectrom. 2000,11, 78-82. 

Figure 4.10. Proteomic analysis by SILAC. Proteomic analysis by SILAC or stable isotope labeling of amino acids in cell culture utilize de novo metabolic incorporation of stable-isotope-labeled amino acids during protein synthesis. Cells can be cultured with various combinations of stable-isotope-labeled amino acids such as lysine or arginine. Tyrosine has been used in phosphoprotein studies of tyrosine residues. About five or six cell divisions are needed for complete labeling of proteins in cell cultures prior to experimentation. Labeled cells from control and treatment(s) lysates are combined and digested. Quantitation and identification are performed by LC-MS/MS.

The method has been used to prepare isotopically labelled amino acids. While Boc-BMI enolate adds to aldehydes with only moderate diastereoselectivity, reduction of the acylation products (5) gives allothreonine derivatives (6). Michael additions to o(,p-unsaturated esters, ketones, and nitro compounds lead to products of type (7) and (8) (for a general discussion see Suzuki and Seebach ). 

Figure 16.5 The dimer structure of PC-1 (PDB code 1ZY6) in POPC bilayers as determined by solid-state NMR. Isotopically labeled amino acids are shown in stick format Phe 12 was labeled with 19F, Cyst2 with 15N and13C, and Vail6 with 13C. Inter- and intramolecular distances were used to determine the relative position of the two monomers. See color plate 16.5.

This applies especially to substances such as the essential oils or lignin. The comparatively small amount of tvork so far done on the biogenesis of alkaloids from isotopically labeled amino acids suggests that theories of biogenesis of alkaloids from amino acids are probably justified. 

Simpson TJ, Willis CL (1996) Enzymatic and chemical methods for the enantioselective synthesis of isotopically labelled amino acids and metabolic intermediates. Proceedings of the Chiral Europe 96 Symposium, Strasbourg, p 49... 

Feeding experiment. Feeding procedures were essentially same as previously reported. Briefly, the stable isotope labeled amino acids and tryptamine were gelled by agarose (Type VII Sigma) and the gel was fed to the specimens in a small dish. After feeding over 10 days, Cypridina luciferin was extracted from 4 animals with ethanol and the extracts were served to LC/ESI-TOF-MS analyses. LC/ESI-TOF-MS. LC/ESI-TOF-MS was performed with an Agilent 1100 HPLC... 

Proton ENDOR is a useful probe of the origin of the EPR signals from organic-based radicals. In a series of cw ENDOR studies including samples with isotopically labeled amino acids, proton ENDOR was used to identify the amino acid radical in cytochrome-c peroxidase (CcP) as a tryptophan species. Similarly, isotopic labeling was used to identify the tyrosyl... 

The proteins of the body are continually being replaced and resynthesized from the amino acids available to the body. The use of isotopically labeled amino acids has made possible studies of the average lifetimes of amino acids as constituents in proteins—that is, the time it takes the body to replace a protein in a tissue. Considering that this process must be extremely complex, replacement is very rapid. Only minutes after radioactive amino acids are injected into animals, radioactive protein can be found. Although... 

The analysis of amino acids by GC-MS of suitable volatile derivatives is a well-established technique. Despite the availability of amino acid analyzers, GC-MS is still occasionally used because it is useful for qualitative and quantitative analysis of unusual (i.e., nonprotein) amino acids. GC-MS is also valuable for conducting studies of the racemization of amino acids during cooking or food processing, when used in conjunction with chiral GC columns and deuterium labeling. GC-MS methods are also often used for determining stable isotope labeled amino acids in nutritional, metabolic studies. ESI LC-MS may also be used for determining amino acids. 

Isotopically labeled amino acids have been administered to excised plant parts. As is true for cyanogenic glycosides, incorporation of administered amino acid precursors into glucosinolates is often extremely high (up to 40%) (Larsen, 1981). In one study, plants were fed amino acids, and biosyn-... 

The in-vivo metaboHc labehng of proteins is achieved by growing cells in the presence of N-precursors [65, 66], or by the addition of isotope-labeled amino acids to the medium. The latter method is known as Stable Isotope Labehng by Amino Acids in CeU Culture (SILAC) [67, 68], and it provides the means for comparative analysis of protein abundance and post-translational modifications by MALDI-MS [69]. Commercially available alkylating reagents include ICAT (Cys)... 

Previous studies have determined which amino acids are incoqx rated into thiostrepton (55) and nosiheptide (56) by feeding isotopically labeled amino acids to the producing... 

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