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.

Comparison between collision induced dissociation of electrosprayed protonated peptides in the up-front source region and in a low-energy collision cell.

A systematic comparison between the up-front Collision induced dissociation (CID) mass spectra and low-energy CID tandem mass spectra from twenty-one singly and/or doubly charged peptides has been made. CID spectra of the peptides were recorded at different electrode voltages in the up-front source region of a single quadrupole instrument and different collision energies in the collision cell of a tandem quadrupole instrument. It was observed that up-front CID and low-energy CID yielded comparable product ion spectra from protonated peptides, and that the instrumental settings necessary for obtaining comparable CID mass spectra from the two methods are correlated.

Validated method for the determination of idazoxan in human plasma by liquid chromatography with tandem mass spectrometric detection.

A liquid chromatography-tandem mass spectrometry method for the determination of idazoxan in human (heparin) plasma is presented, which was developed and validated using 500 microl of sample. Sample preparation consisted of the addition of fluoroidazoxan as the internal standard, extraction at alkaline conditions into tert.-butyl methyl ether, followed by centrifugation, evaporation of the solvent and reconstitution in methanol. After a short chromatographic run, detection took place by ionspray tandem mass spectrometry in positive ion mode. Validation results on linearity, specificity, accuracy, precision and stability, as well as application of the method to samples from a clinical trial, are shown. The validated calibration range is from 0.300 to 100 ng/ml, with accuracy (bias) and precision (coefficient of variation) being below 15% at all levels. A sample throughput of, typically, 150 per day can be achieved.

Statistical analysis of mass spectral data obtained from singly protonated peptides under high-energy collision-induced dissociation conditions.

A statistical study of the fragmentation behaviour of 138 model peptides, containing 3-9 amino acid residues (n = 3-9) under high-energy collision conditions is presented. The aim was to identify characteristic patterns of ions in the spectra of peptides which can be translated into general rules to be used in the spectral interpretation and provide a better insight into their fragmentation behaviour. It was found that both number and nature of the amino acids are important factors directing the fragmentation behaviour. The spectra of tri- and tetrapeptides exhibit a comparable probability for the formation of B2- and Y"n-2 ions, whereas larger peptides show a preference for the formation of Bn-1 ions. This generally observed fragmentation pattern of peptides is changed significantly when basic amino acid residues (Arg, Lys and His) and/or Pro are present Arginine appears to have the most pronounced influence on the fragmentation behaviour and overrules that of the other amino acid residues.

Identification of a new genetic variant of bovine beta-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis.

Various components of the beta-casein fraction from bovine milk were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). They included the genetic variants beta A1, beta A2, beta A3, and an unknown component previously denoted beta X [S. Visser et al., J. Chromatogr. 548 (1991) 361-370]. Tryptic digests of these components were compared by RP-HPLC and most peaks were analysed by mass spectrometry (MS). The tryptic map of beta X was closest to that of beta A1, but with a few mutually different peak components. Electrospray ionisation MS revealed that in the beta X map these components had relative molecular masses of 16 higher than the corresponding ones in the beta A1 map. The main differential peaks represented the 114-169 fragments of beta A1 and beta X, respectively, which were both purified and then cleaved with cyanogen bromide. In the resulting mixtures, each of which contained three fragments, the corresponding peptides representing the 145-156 sequence showed the 16 relative molecular mass difference. In beta X this sequence contained a Leu residue at position 152 instead of the Pro-152 in beta A1, as established by fast-atom bombardment MS-MS. The Leu could be discriminated from an Ile residue by the presence of a side-chain-specific, D-type fragment ion in the MS-MS spectrum of the beta X CNBr peptide. The sequence of the two homologous 145-156 fragments was confirmed by regular amino acid sequence analysis. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named beta-casein F-5P.

Identification of the active site histidine in Staphylococcus hyicus lipase using chemical modification and mass spectrometry.

Staphylococcus hyicus lipase is a serine hydrolase. In order to identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase.

Rapid identification of specific mutations in the sequence of an enzyme variant produced by protein engineering using high-performance liquid chromatographic/fast atom bombardment mass spectrometric techniques.

Unknown, specific mutations in the sequence of an enzyme variant (a Bacillus subtilisin protease) produced by protein engineering were identified using High-performance Liquid Chromatographic/Fast Atom Bombardment Mass Spectrometric (HPLC/FAB MS) techniques. The variant and the highly homologous wild-type enzyme were treated with CNBr followed by tryptic digestion. The resulting peptides were analysed using HPLC/frit FAB MS. The peptides with molecular masses beyond the range of the HPLC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS. This procedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference.

Fast atom bombardment mass spectrometry of some lantibiotics.

Four lantibiotics namely epidermin, gallidermin, lanthiopeptin and mersacidin, have been studied by fast atom bombardment mass spectrometry. The molecular ion clusters of these compounds can be detected with reasonable abundance. The low-mass regions of the spectra show the presence of ions characteristic of the amino acids in the peptides. The mass distribution of the sequence ions provides information about the location of sulphur bridges the occurrence of which is a common feature of these kinds of molecules. The two isomeric compounds epidermin and gallidermin differ only in a leucine/isoleucine exchange at position 6. These two compounds can be distinguished on the basis of the tandem mass spectrum of m/z 86, the immonium ion of leucine and isoleucine.

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine kappa-casein.

The specificity of the cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine kappa-casein was studied. A 4-h digest (pH 6.2, 15 degrees C) of kappa-casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1-160, 1-151, 1-95 and 1-79 of kappa-casein, whereas the main LMM products found were the 161-169 and 152-160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160-161 and 151-152 peptide bonds. Two minor LMM products were identified as the fragments 96-104 and 103-106, indicating additional cleavage at positions 102-103, 104-105 and 106-107 of the sequence. Also several peptide bonds within the 161-169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococcal CEPI, CEPIII and several mixed-type proteinases all act on the peptide bonds at positions 79-80 and 95-96.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid analysis of enzymatic digests of a bacterial protease of the subtilisin type and a "bio-engineered" variant by high-performance liquid chromatography-frit fast atom bombardment mass spectrometry.

Amino acid sequencing of a subtilisin-type bacterial protease and a bio-engineered variant was carried out by investigating various enzymatic digests using HPLC-frit fast atom bombardment MS methods. The fast atom bombardment mass spectral data allowed rapid identification of the enzymatically generated peptides and differentiation between both proteins. The feasibility of determining the positions and nature of mutations in the amino acid sequence depends mainly on the size of the peptides containing the modifications.

Sequence-ion studies in peptides: the generation of C" ions.

The mechanism for the formation of C"-type ions from protonated peptides, produced under conditions of fast-atom bombardment and collisional activation was investigated. Comparison of the tandem mass spectra of the [M + H]+ ions of a model peptide and the corresponding [Md + D]+ ions, in which all exchangeable hydrogens are replaced with deuterium, revealed that neither the carboxylic hydrogen nor a hydrogen from a nitrogen atom is involved in the process of migration of a hydrogen which leads to the formation of C"n-type ions. The most feasible position from which the transferred hydrogen originates is that at the first C-atom in the side-chain of the adjacent amino acid.