A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds. We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.

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The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipidbinding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/b/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1-and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipidbinding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.Keywords: crystal structure; flavohemoprotein; globin; phospholipid; protein engineering.Flavohemoprotein is a monomeric protein with a molecular mass of 43 kDa, which binds one heme (Fe-protoporphyrine IX) and one FAD as prostethic groups and NADH as a cofactor. This protein has been identified and characterized in an expanding number of prokaryotic and eukaryotic organisms, comprising Flavohemoprotein was originally isolated from the gramnegative bacterium A. eutrophus (denoted as FHP) [11] and characterized with respect to its spectral properties [12], its primary structure [1] and its crystal structure [13]. According to a 0.175-nm structure, FHP can be subdivided into three domains. The N-terminal 147 residues comprise the well-known globin fold[14] composed of seven a helices forming a hydrophobic crevice, where the heme molecule is embedded. The FAD-binding domain (105 residues) basically consists of a six-stranded antiparallel b barrel surrounded by a helix and an irregular peptide segment. The noncovalently bound FAD binds roughly to the face of the b sheet. The architecture of the NAD-binding domain (138 residues) corresponds to a Rossmann fold composed of a five-stranded parallel b sheet flanked by two helices and an irregular structural element. The binding mode of NADH is not yet established. The FAD-binding and NAD-binding domains are fused to the so-called oxidoreductase module, which structurally belongs to the ferredoxin reductase family [15]. FHP is therefore composed of two essentially independent globin and oxidoreductase modules.Lipid-binding protei...

RationaleThe fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off‐line preparation to reveal this information. An automated position‐specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ 13CCARBOXYL values). This automation makes PSIA measurements easier and routine.MethodsAn existing high‐performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post‐column derivatisation unit between the HPLC system and the interface. The post‐column reaction was optimised to yield CO2 from the carboxyl groups of amino acids by reaction with ninhydrin.ResultsThe methodology described produced δ 13CCARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ 13CCARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ 13CCARBOXYL values of a number of internationally available amino acid reference materials.ConclusionsThe PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole‐molecule level. When combined with on‐line chromatographic separation or off‐line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position‐specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.