On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. I. Determination of clenbuterol in urine.

The potential of the direct coupling of solid-phase extraction (SPE) with mass spectrometry (MS) for the analysis of biological samples is demonstrated. For SPE a cartridge exchanger is used and the eluate is directly introduced into the mass spectrometer. This system has been investigated for the determination of clenbuterol in urine. With mixed-mode cartridges, a considerable ion suppression has been obtained. The mass spectrum at the elution time of clenbuterol is dominated by that of creatinine and adduct formation of clenbuterol and creatinine has been observed. The whole procedure including injection of 1 ml urine, washing and desorption has been developed with cartridges containing 8-microm C18-bonded silica. If only a single MS step is used, the selectivity and, therefore, the sensitivity are insufficient. The detection limit is about 100 ng/ml. However, with atmospheric pressure chemical ionisation and the tandem MS mode the detection limit has been decreased to about 2 ng/ml and the ion suppression is only about 10%. For the electrospray ionisation the detection limit is about 10-times higher and the ion suppression is less favourable. The repeatability for the SPE-MS-MS procedure was 6.5% at 10 ng/ml (n=5) and the difference between the response factors at 10 ng/ml and 100 ng/ml was only 2.5%. The MS behaviour of clenbuterol and the matrix under the present conditions is discussed.

Reconstruction of the proteolytic pathway for use of beta-casein by Lactococcus lactis.

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.

Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase.

Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. Previous work has shown that Asp124, which is located close to the internal substrate-binding cavity, carries out a nucleophilic attack on the C-alpha of the alkylhalide, displacing the halogen. The resulting alkyl-enzyme intermediate is subsequently hydrolyzed. In order to study the role of His289 in the hydrolysis of the intermediate, a His289-->Gln mutant was constructed by site-directed mutagenesis. The purified mutant enzyme was not catalytically active with haloalkanes, but a halide burst stoichiometric to the amount of enzyme was observed with 1,2-dibromoethane. Using ion spray mass spectrometry, accumulation of the covalent alkyl-enzyme and binding of the alkyl moiety of the substrate to an Asp124-containing tryptic peptide were shown. Fluorescence-quenching experiments indicated that halide ions are strongly bound by the alkyl-enzyme but not by the substrate-free enzyme. The results show that His289 is the base catalyst for the dealkylation of the covalent intermediate, but that it is not essential for the initial nucleophilic attack of Asp124 on the C-1 atom of the haloalkane. Furthermore, the halide ion that is released in the first step probably leaves the active site only after hydrolysis of the alkyl-enzyme.

Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences.

The primary structure of pseudo-hevein, a minor hevein component from the latex of the rubber tree, Hevea brasiliensis, was determined. Six differences with the sequence of the major hevein component were found, one of which is a replacement of tryptophan by tyrosine in the carbohydrate binding region of the molecule. Analysis by ion-spray mass spectrometry showed that pseudo-hevein has a heterogeneous C-terminal extension of several glycine residues and that hevein itself also contains minor components with additional C-terminal amino-acid residues. A seventh difference between the two sequences occurs in these extensions.

Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase.

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. The X-ray structure suggests that Asp124, which is located close to an internal cavity, carries out a nucleophilic attack on the C alpha of the substrate, releasing the halogen. To study the mechanism of hydrolysis, this aspartate residue was mutated to alanine, glycine, or glutamate. The mutant enzymes showed no activity toward 1,2-dichloroethane and 1,2-dibromoethane. Incubation of purified wild-type dehalogenase with 1,2-dichloroethane in the presence of H2(18)O resulted in the incorporation of 18O in 2-chloroethanol and in the carboxylate group of Asp124. This shows that the reaction proceeds by covalent catalysis with the formation of an alkyl-enzyme intermediate that is hydrolyzed by attack of solvent water on the carbonyl carbon of Asp124. On the basis of amino acid sequence similarity between haloalkane dehalogenase and epoxide hydrolases, it is proposed that a conserved aspartate residue is also involved in covalent catalysis by the latter enzymes.