The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding.

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.

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.

Sodium-cationized oligosaccharides do not appear to undergo 'internal residue loss' rearrangement processes on tandem mass spectrometry.

The phenomenon of 'internal residue loss' of protonated native- and per-O-methylated oligosaccharides has recently been described as occurring on high-energy collision conditions. Awareness of this phenomenon in the mass spectrometric analysis of oligosaccharides is of great importance since the rearrangement ions produced by this process may complicate monosaccharide sequence assignment. In this research, oligosaccharides having N-acetyl-glucosamine residues as the reducing or non-reducing terminal residue have been included in our MS/MS analyses in order to try to better understand the factors that influence 'internal residue loss'. Native and per-O-methylated compounds were submitted to positive and negative MS/MS, selecting protonated, sodium-cationized, or de-protonated pseudomolecular ions as precursors. High- and low-energy collision induced dissociation tandem mass spectrometry experiments were performed using a four sector instrument and a hybrid quadrupole time-of-flight mass spectrometer respectively. The phenomenon of 'internal residue loss' was not observed on either high- or low-energy CID-MS/MS when sodium-cationized precursor ions of either native or per-O-methylated oligosaccharides were examined. Similarly, MS/MS analysis performed in the negative ionization mode also failed to generate ions resulting from 'internal residue loss'. This combination of experiments therefore offers a way to be sure whether ions observed in the tandem mass spectra of protonated native or per-O-methylated oligosaccharides originate from 'internal residue loss' or from direct glycosidic linkage fragmentation.

Assessment of nitric oxide production by measurement of [15N]citrulline enrichment in human plasma using high-performance liquid chromatography-mass spectrometry.

Nitric oxide (NO) is formed by a class of NO synthases (NOS), which convert arginine into citrulline. A decreased in vivo NO availability can be the result of an increased NO inactivation or a decreased NO production. The latter can be assessed by measurement of isotopic enrichment of plasma citrulline during infusion of isotopically labeled arginine. The potential of high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) to determine enrichments of [15N2]arginine and [15N]-citrulline in plasma during infusion of [15N2]arginine in humans was investigated. Two types of MS instruments were evaluated: a sector-type mass spectrometer equipped with a frit fast-atom bombardment (FAB) interface and a quadrupole instrument with electrospray ionization (ESI). FAB-MS appeared to be unsuitable for determination of isotope ratios, because background ions influenced the observed isotope ratio in an unpredictable way. In combination with either off- or on-line reversed-phase HPLC, ESI-MS proved to be a more reliable technique. However, the amount of material that is introduced in the mass spectrometer is critical and should be carefully controlled. During infusion of [15N2]arginine in 14 healthy subjects, a mean arginine-to-citrulline conversion rate of 0.22 +/- 0.07 (SD) mumol.kg-1.h-1 was found. In 4 subjects who received an intravenous infusion with the NOS antagonist L-NMMA, the conversion rate decreased from 0.30 +/- 0.14 to 0.10 +/- 0.06 mumol.kg-1.h-1. It is concluded that ESI-MS in combination with HPLC can be successfully applied for determination of arginine and citrulline enrichments in plasma, thus providing a useful tool for assessment of in vivo NO production.

Preparation of site-specific isotopically labelled zervamicins, the antibiotic peptaibols produced by Emericellopsis salmosynnemata.

A simple procedure for the preparation of the specifically labelled peptide antibiotic zervamicins IC, IIA and IIB has been developed. The zervamicin molecules are labelled with stable isotopes by culturing the Emericellopsis salmosynnemata on a well-defined synthetic medium containing the highly isotopically enriched amino acid. To obtain the peptide with the specifically and highly enriched amino acid residue, precautions have been taken to prevent any de novo biosynthesis of the particular amino acid from unlabelled precursors. The enrichment of the labelled peptide is determined by mass spectrometric analysis. Following this method we have incorporated [2',4',5',6',7'-2H5]-L-Trp-1, [1'-15N]-L-Trp-1 and [2',3',4',5',6'-2H5]-L-Phl-16 into zervamicins IC, IIA and IIB on the preparative scale and without scrambling of the label. Thus, using the procedures described, isotopically labelled zervamicins can be prepared, allowing them to be studied by solid-state NMR.

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.

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Phosphatidylcholine transfer protein from bovine liver contains highly unsaturated phosphatidylcholine species.

The phosphatidylcholine transfer protein (PC-TP) from bovine liver contains one molecule of non-covalently bound PC. In order to gain more insight into the physiological function of PC-TP, PC was extracted from bovine liver PC-TP and its molecular species composition identified by fast atom bombardment mass spectrometry. The prevailing molecular species were C18:0/C18:1-, C18:0/C18:2-, C18:0/C20:4-, C18:0/20:5- and C18:0/C22:5-PC accounting for 85% of the PC species present. This molecular species composition is not representative for what is present in bovine liver where these species account for 43% of the total PC content [Montfoort et al. (1971) Biochim. Biophys. Acta 231, 335-342]. Another striking observation is that PC species carrying a palmitoyl chain at the sn-1 position are nearly absent, despite these species being abundantly present in bovine liver. This study suggests that PC-TP could play a role in the metabolism of highly unsaturated, stearoyl-containing PC species.

Comparing mass spectrometric characteristics of peptides and peptoids.

The collision-induced dissociation (CID) spectra of the [M+H]+ ions of a pentapeptide and the corresponding peptoid and retropeptoid have been compared. The spectra of the peptide and peptoid both exhibit B- and Y"-type sequence ions at identical m/z values. In contrast to the peptide, the [M+H]+ ion of the peptoid and all sequence ions containing an N-substituted glycine derivative corresponding to a tyrosine amino acid residue can easily lose a C7H6O molecule in a charge-remote fragmentation process. The presence of N-substituted glycine residues in a peptoid is further apparent from the presence of N-substituted immonium ions, which differ significantly in their fragmentation behaviour from the corresponding immonium ions observed in the spectra of common oligopeptides. Loss of the CH2 = NH imine molecule is the dominant fragmentation reaction in the CID spectra of all peptoid immonium ions investigated in this study. The elimination of the CH = NH2 ylide analogue from common peptide immonium ions is energetically less favourable as shown by ab initio calculations. The relative heat of formation of the CH = NH2 ylide neutral appeared to be 168 kJ mol-1 more than that of the CH2 = NH imine molecule.

Identification of oxidized methionine in peptides.

The positive- and negative-ion collision-induced dissociation spectra of peptides containing methionine, methionine sulphoxide and methionine sulphone have been studied. Characteristic fragmentations were identified and evaluated as possible indicators for the presence of oxidized methionine residues in peptides. It was found that the elimination of CH3SOH (-64 u) from [M + H]+ is unique for peptides that contain methionine sulphoxide. Sequence ions containing the oxidized methionine undergo the same elimination, allowing unambiguous sequence determination. Methionine sulphone exhibits an analogous elimination of CH3SO2H (-80 u) from the protonated molecule, but not from sequence ions.

Tandem mass spectrometry and nuclear magnetic resonance spectroscopy studies of Candida bombicola sophorolipids and product formed on hydrolysis by cutinase.

Natural mixtures of sophorolipids produced by the yeast Candida bombicola have been analyzed by fast atom bombardment (FAB)-MS and collision-induced dissociation (CID)-MS. Some pure components have been analysed by two-dimensional NMR spectroscopy. The presence of acidic, lactonic, and O-acetylated forms and the position of double bonds in the fatty acid part of these glycolipids can be easily inferred from positive and negative ion FAB-mass spectra. Details about position of O-acetylation can be obtained from CID mass spectra of [M+H]+ and [M-H]- ions and from the NMR spectra. Differences in CID fragmentation between protonated and sodiated molecular ions are discussed in detail. Enzymatic hydrolysis of 6',6"-di-O-acetyl sophorolipid lactone by cutinase from Fusarium solani results specifically in the removal of the 6'-O-acetyl group, whereas the 6"-O-acetyl and lactone group are resistant. This specificity is explained from a three-dimensional model of the sophorolipid generated on the basis of the short 1H,1H distances as inferred from the NMR (ROESY) spectra.

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)

High-performance thin-layer chromatography/fast atom bombardment (tandem) mass spectrometry of Pseudomonas rhamnolipids.

Several rhamnolipid preparations from Pseudomonas strains were studied by thin-layer chromatography/fast atom bombardment (TLC/FAB) mass spectrometry and TLC/FAB tandem mass spectrometry (MS/MS). The preparations were separated with normal-phase (Silica 60) and reversed-phase (RP-8) chromatography. Silica 60 plates appeared to be very useful in the separation of rhamnolipids according to the number of monosaccharide residues present. Spectra which show characteristic fragment ions could be obtained from components of mixtures with a total sample size of less than 200 ng. Chromatography on RP-8 plates gave a good separation of the rhamnolipids based on the length of the fatty acid alkyl chain. MS/MS of the sodium cationized molecules gave information about the sequence of the building blocks. Particularly, heterogeneity in beta-hydroxy fatty acid composition was determined for the principal as well as minor components present in natural rhamnolipid mixtures.

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.

Determination of linkage positions in peracetylated (methyl) xylo-oligosaccharides with fast atom bombardment mass spectrometry.

Distinction between the linkage types 1-->2, 1-->3 and 1-->4 of xylobioses can be achieved on the basis of the unimolecular decomposition spectra of the oxonium ions of the per-O-acetylated methyl glycosides. The spectra of the oxonium ions of various unbranched xylotri-, tetra- and pentaoses allow determination of the linkage position between the xylose residues. This indicates that in unbranched peracetylated xylo-oligosaccharides the linkage between the xylose residues at the non-reducing end can be determined.

Mass spectrometric discrimination between per-O-acetylated aldopentoses applied to an oligosaccharide.

Unimolecular decomposition spectra of oxonium ions from peracetylated pentoses can be used for identification purposes. The fast-atom bombardment mass spectra of an oligosaccharide with a pentose residue at the non-reducing terminus also contain this oxonium ion, which allows identification of this monosaccharide residue by a tandem mass spectrometry experiment. The applicability of this method is demonstrated using a xylopentaose.

Structure identification of natural rhamnolipid mixtures by fast atom bombardment tandem mass spectrometry.

Two rhamnobiose-lipid preparations have been studied by fast atom bombardment (FAB) tandem mass spectrometry. The principal rhamnobiose-lipids contain the beta-hydroxydecanoyl-beta-hydroxydecanoate Rha-Rha-C10-C10 and the beta-hydroxytetradecanoyl-beta-hydroxytetradecanoate Rha-Rha-C14-C14. Both preparations contain minor components which are heterogenous in beta-hydroxy fatty acid composition. FAB ionization of rhamnobiose-lipids in the presence of Na+ shows the formation of both [M + Na]+, [M +2Na-H]+, [M + 3Na-2H]+ and [M - H]- ions. Tandem mass spectrometry of the [M + 2Na-H]+ and [M - H]- ions give information about the sequence of the building blocks. Particularly, heterogeneity in beta-hydroxy fatty acid composition is determined for the principal components and all the minor components present in the preparations.

Fast atom bombardment mass spectrometry of sucrose monocaprate and sucrose monolaurate.

Fast Atom Bombardment (FAB) ionization of sucrose monocaprate and sucrose monolaurate in the presence of Na+ ions shows the formation of both [M+Na]+ and [M - H]- ions. The [M+Na]+ ions undergo charge-remote fragmentations when collisionally activated at high translational energy. These charge-remote fragmentations are useful for the structural determination of the acyclic part of the glycolipid. In the negative ion mode both sucrose esters yield anions which can be attributed to the saccharide and lipid components of these glycolipids. Structural characterization of the fatty acid can be achieved as the released carboxylate anions undergo charge-remote fragmentations that are consistent with the ion chemistry of [M - H]- anions from FAB-ionized free fatty acids.