Isotopomers peptides

Coupling constants are routinely used to determine the side-chain conformation of amino acids in peptides and proteins. Whereas proteins nowadays are almost exclusively studied as C- and N-labeled isotopomers, peptides usually have these isotopes in natural abundance, i.e. the magnetically active heteronuclei are highly diluted. Most amino acids contain a methylene group at the ji-position for which the X angle is determined by the conformation of the Ca—Cp bond. Two vicinal Jhh coupling constants can be measured Ha to and H to Usually... 

Example Peptides often contain sulfur from cysteine. Provided there are at least two cysteines in the peptide molecule, the sulfur can be incorporated as thiol group (SH, reduced) or sulfur bridge (S-S, oxidized). Often, both forms are contained in the same sample. At ultrahigh-resolution, the contributions of these compositions to the same nominal m/z can be distinguished. The ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) FT-ICR mass spectrum of native and reduced [D-Pen jenkephalin gives an example of such a separation (Fig. 3.25). [39] The left expanded view shows fully resolved peaks due to and C2 isotopomers of the native and the all- C peak of the reduced compound at m/z 648. The right expansion reveals the peak of the native plus the... 

Multidimensional and dual frequency infrared analogues of NMR Applications to peptides and isotopomers of secondary structures... 

Amide III VCD from aqueous solution was published in 1987 [31], and normal coordinate analyses of simple peptides and a number of isotopomers were carried out to define the exact nature of the amide III vibration [32]. Recently, we have reported a detailed comparison of computational and experimental VCD results in the amide I region [33]. Keiderling has pushed the frontiers toward collecting VCD data on a number of proteins, and interpreting the data, via factor analysis, in terms of percentages of the common secondary structures [34,35]. A number of excellent reviews, summarizing the progress in peptide VCD in the 1985-1991 time span, have appeared [36,37],... 

While empirical rules would fail to yield a correct conformational interpretation of the amide I spectrum, normal mode calculations using the SQM method clearly eliminate several possibilities and put forward a preferred structure for this peptide in water [70S], This approach can be extended to much larger peptides having stable secondary structures. We have collected spectra of several isotopomers of the 23-residue peptide magainin F. We observe in the difference spectra that the amide I bands corresponding to specific amino acids are much narrower than they are in the short peptides and clearly identify the amide I frequencies of these groups. 

Different 2H-, 13C- and/or 15N isotopomers of L-serine, [(S)-2-amino-3-hydroxypro-panoic acid], 95, required for studies of aminoacid metabolism and for studies of peptide and protein structure and dynamics, have been biosynthesized stereoselectively84 using the serine-type methylotrophic bacterium, Methylobacteri extorquens AMI, which contains85 large amounts of the enzymes methanol dehydrogenase and hydroxymethyl-transferase (equation 39). 

Relative protein quantitation is the basis of all types of differential proteome analyses. In the 2D-gel approach protein staining with either visible or fluorescent dyes provides a reliable and sensitive method to detect changes in protein expression or isoform abundance. In the multidimensional LC approach quantitation relies mostly on stable isotope labeling and ratios between light and heavy isotopomers are determined by MS or MS/MS at the peptide level. Labeling can be performed on the protein level by... 

H, C, Si, and using two cardinal types of pulse sequences based on the HNCO and HNCA experiments which constitute standard schemes routinely employed for structure elucidation of peptides and proteins. However, whereas work on biomolecules is generally carried out with uniformly and labelled molecules, these studies were performed in natural abundance of isotopes, which required some adaptations of the experiments in order to improve the suppression of signals from isotopomers with magnetically inactive and Si nuclei. 

Polyglycine, H(-CO-CH2-NH)n-H, is the simplest polypeptide. It has two solid state structures. Form I consists of chains of polymer that are hydrogen-bonded into two-dimensional sheets. This form (including selectively deuterated isotopomers) has been studied by INS [53-55]. The spectra were interpreted similarly to those of N-methylacetamide. Reexamination of the spectra with periodic-DFT calculations is necessary. Acetanilide, C6H5-NH-C(=0)-CH3, forms hydrogen-bonded chains similar to N-methylacetamide and as such is a potential model for phenyl substituted peptides. However, the interest in acetanilide is that the... 

Acetanilide, and some of its isotopomers, have been studied by INS spectroscopy [56-58]. The dispersion curves of the fully deuterated material have been measured by coherent INS [59]. A comprehensive analysis of acetanilide in the solid state was carried out with molecular dynamics simulations [57]. This includes all the lattice modes, as shown in Fig. 10.27 The simulations suggested that the barrier to the methyl torsion was enhanced when the peptide group is hydrogen-bonded and that this was a through-bond polarization effect. The methyl torsion was... 

One method for temporal precnrsor isolation is stored waveform inverse Fourier transform (SWIFT) [40]. In this method, the desired freqnency domain profile (all frequencies except that of the ion of interest) is inversely Fonrier transformed to a time domain waveform. This waveform is then applied to the excite electrodes in the ICR cell and, thns, the precursor ions are isolated in the cell. An alternative techniqne for in-cell isolation is correlated sweep excitation (COSE) [41], also known as correlated harmonic excitation fields (CHEF) [42]. This method involves application of radiofrequency pulses to the excite electrodes. The technique correlates the duration and frequency of the RF-pulses with those appropriate to the ions to be isolated. Both SWIFT and COSE are capable of isolating single isotopomers in peptide and protein ions [43-45]. 

Gly-Tyr was synthesized as both the [ C]-amido (90 atom % enrichment and amido-[ C, N] (90 atom % enrichment, respectively) isotopomers by known methods. CPA, purchased from Sigma as a suspension in toluene, was crosslinked and soaked with Gly-Tyr as described previously, and the washed, dried crystals examined by solid-state NMR. The C CPMAS NMR spectrum of the C-enriched Gly-Tyr (Fig. 12) displays residual C- N quadrupolar coupling (8 = 176 ppm). However, the C CPMAS difference spectrum of CPA and the CPA/Gly-Tyr complex (Fig. 12) displays a single resonance (5 = 178 ppm, LW /2 60 Hz) revealing that, under the experimental conditions, the peptide bond of this slow reacting substrate has been cleaved to the extent of > 90%. 

R 505 D. Seebach, A.K. Beck and DJ. Bierbaum, The World of (3- and y-Peptides Comprised of Homologated Proteinogenic Amino Acids and Other Components , Chem. Bio divers., 2004,1,1111 R 506 D.S. Sem Chemical Proteomics from a Nuclear Magnetic Resonance Spectroscopy Perspective , Expert Rev.Proteom., 2004,1,165 R 507 A.D. Sherry, F.M.H. Jeffrey and C.R. Malloy, Analytical Solutions for C Isotopomer Analysis of Complex Metabolic Conditions Substrate Oxidation, Multiple Pyruvate Cycles, and Gluconeogenesis , Me-tab.Eng., 2004,6,12... 

Derivatization is simply the attachment of a labeled building block to the unlabeled target. Clearly, this produces not the structurally identical isotopomer, but a labeled derivative. Nevertheless, this approach can be useful if the performance of the derivative in the intended studies mimics to an acceptable degree that of the original structure i.e., the changes in the physical or chemical properties incurred in the derivatization do not alter the study parameters that are measured. Derivatization can be advantageous for complex compounds such as proteins, peptides or complex natural products that would otherwise be difficult to label. Preliminary evaluation of the derivative in the intended assay is essential to ensure its suitability. 

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