Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?

A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.

Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli.

1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.

Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi.

Photobacterium leiognathi lumazine protein has been expressed in Escherichia coli in high yield, 30 mg/l. The cloned gene was one previously reported by Illarionov (EMBL X56534), that had a similar sequence and was located in the same position as the lumazine protein gene in P. phosphoreum. This gene was placed downstream of the T7 gene 10 promoter of the plasmid pT7-7. When the E. coli are grown at 37 degrees C the protein accumulates in inclusion bodies but solubilization can be achieved in 6 M urea. By a simple procedure of dialysis in the presence of riboflavin and centrifugation, without any chromatography, the recombinant holoprotein is purified to 95% homogeneity. The spectral properties of this recombinant riboflavin protein are the same as those of a fluorescent riboflavin-bound protein produced by many strains of P. leiognathi. The absorption spectrum has the same maxima, 276, 386, 464 nm, the circular dichroism is also the same, and both absorption spectrum and CD are the same as that of apo-lumazine protein having riboflavin bound. The riboflavin on the recombinant can be easily replaced by 6,7-dimethyl-8-ribityllumazine. The absorption and fluorescence spectra, fluorescence yield, and bioluminescence properties of this rebound protein identify it as authentic lumazine protein.

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas.

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.

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.

Overproduction of the prismane protein from Desulfovibrio desulfuricans ATCC 27774 in Desulfovibrio vulgaris (Hildenborough) and EPR spectroscopy of the [6Fe-6S] cluster in different redox states.

The Desulfovibrio desulfuricans ATCC 27774 prismane protein was isolated from a Desulfovibrio vulgaris (Hildenborough) strain that contained the gene for this protein in expression vector pSUP104. A redox titration demonstrated that the [Fe-S] cluster in this protein may attain four different redox states, indicated as +3, +4, +5 and +6, with midpoint potentials for the transitions of approx. -220, +50/-25 and +370 mV, respectively. EPR spectra of the protein in the various redox states are reminiscent of those of the D. vulgaris prismane protein (Pierik et al. (1992) Eur. J. Biochem. 206, 705-719), but differ in details. In the +5-state, virtually all the iron is in a S = 9/2 spin state, indicative for a cluster that is more complex than common [4Fe-4S] or [2Fe-2S] clusters. Similarity of the EPR spectrum of the protein in the +3-state with those of inorganic [6Fe-6S] model compounds suggests that the cluster in the protein is also [6Fe-6S]. In the +4-state of the protein a broad signal due to an integer-spin system can be detected with normal-mode EPR. A dramatic sharpening-up and increase of intensity of this band (g = 14.7) is observed with parallel-mode EPR. In accordance with the chemically determined iron content of the protein (6.0 +/- 0.45 moles of iron/mole of protein), the spectroscopic data indicate one [6Fe-6S] cluster in this protein. We did not find evidence for a previous claim (Moura et al. (1992) J. Biol. Chem. 267, 4489-4496) that the D. desulfuricans protein contains two [6Fe-6S] clusters.

Mass spectrometric identification of phosphorylated vasostatin II, a chromogranin A-derived protein fragment (1-113).

Vasostatin II, an N-terminal chromogranin A-derived protein (CGA1-113), was purified from bovine chromaffin granule lysate and characterized by electrospray mass spectrometry (ES/MS) as being partially phosphorylated. The phosphorylation site was determined to be at the Ser81 position by mass spectrometric peptide mapping and tandem mass spectrometric analysis. This phosphorylation site is close to the processing site (...QKK78HSS(p)81...) yielding vasostatin I, an N-terminal CGA-derived peptide comprising residues 1-76, suggesting that phosphorylation at Ser81 is involved in the formation of vasostatin I in chromaffin cells.

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry.

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.

The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry.

For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 > m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2'-deoxyguanosine (dGuo) adduct of Benzo[a]pyrene.

The operon for the Fe-hydrogenase in Desulfovibrio vulgaris (Hildenborough): mapping of the transcript and regulation of expression.

The genes for the subunits of the Fe-only hydrogenase from Desulfovibrio vulgaris are transcribed as a 1.9 kb mRNA; the operon contains no other genes besides those encoding the two subunits. The transcriptional start site of the operon was mapped. Determination of hydrogenase activity and hydrogenase mRNA levels indicates a growth-phase dependent regulation of hydrogenase expression at transcriptional level. However, it has not yet been possible to localize the sequences required for regulation and expression of the genes.

hyd gamma, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase.

Downstream of the genes for the structural alpha and beta subunits of the periplasmic Desulfovibrio vulgaris (Hildenborough) hydrogenase a DNA fragment was detected with sequence homology to these genes. This fragment was cloned in Escherichia coli and the nucleotide sequence was determined. A gene was detected on the fragment with coding capacity for a 65.8 kDa polypeptide, hyd gamma. The central part of hyd gamma has an unusually high degree of homology with the alpha subunit and the C-terminal part has similarity with the beta subunit. These results strongly suggest that the three genes for hyd gamma and the alpha and beta subunits derive from one common ancestor gene. We succeeded in the identification of the translational product of this gene in E. coli, but were unable to determine the function of hyd gamma after expression in E. coli.

Equilenin-2'-deoxynucleoside adducts: analysis with nano-liquid chromatography coupled to nano-electrospray tandem mass spectrometry.

The interaction of 4-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupling of miniaturized liquid chromatography (LC) to electrospray tandem mass spectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reaction of 4-hydroxyequilenin with the main 2'-deoxynucleosides were separated on a Hypersyl C(18) BDS nano-HPLC column (15 cm x 75 microm i.d.) at a flow-rate of 300 nl min(-1) using gradient elution with CH(3)OH--0.2% CH(3)COOH in H(2)O. The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution low-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts were found. The nano-HPLC column-switching ES-MS system was tested for its sensitivity on a triple-quadrupole instrument, and detection limits down to 197 fg in the single reaction monitoring mode were obtained for semi-preparatively isolated equilenin--2'-deoxyguanosine adduct.

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.

Study of the enzymatic degradation of vasostatin I and II and their precursor chromogranin A by dipeptidyl peptidase IV using high-performance liquid chromatography/electrospray mass spectrometry.

The interaction of dipeptidyl peptidase IV with structurally related proteins differing in chain length, namely vasostatin I and II and their precursor protein chromogranin A, was examined using high-performance liquid chromatography in combination with electrospray mass spectrometry. Suitable analytical procedures were developed involving the use of reversed-phase high-performance liquid chromatography for purification of the enzymatic degradation products and a peptide mapping procedure for evaluating the enzymatic degradation of the large precursor protein chromogranin A. While vasostatin I was found to be a substrate for dipeptidyl peptidase IV, no N-terminal cleavage of Leu-Pro could be noted for chromogranin A. With respect to vasostatin II, N-terminal degradation was only observed after degradation in the C-terminal domain to proteins containing < or = 78 amino acids. The specificity of the N-terminal release of Leu-Pro was proved by addition of a DPP IV specific inhibitor.

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.

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.