A multiple prognostic index predictive of disease outcome after irradiation for clinically localized prostate carcinoma

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI LC-MS-MS) with daughter ion scan. All TCs gave [M+H-NH3]+ and [M+H-NH3-H2O]+ as the product ions, except for DC when [M+H]+ was selected as the precursor ion. The combination of C18 cartridge clean-up and the present ESI LC-MS-MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry

We developed a method to elucidate the complete structure of triacylglycerols by means of high-energy collisional activation tandem mass spectrometry (MS/MS). Both ESI- and FAB-produced [M + NH4]+ and [M + met.]+ ions (where met. = Li, Na, and Cs) of triacylglycerols undergo charge-remote and charge-driven fragmentations. We emphasize the study of fragment ions from ESI-produced [M + NH4]+ and [M + Na]+ ions and FAB-produced [M + Na]+ ions. ESI-produced [M + NH4]+ ions fragment to produce four types of ions, [M + NH4 - RnCOONH4]+, [RnCO + 128]+, [RnCO + 74]+, and RnCO+ ions, from which the carbon number and the degree of unsaturation of each acyl group are obtained. In addition, three series of ions are produced by charge-remote fragmentations (CRFs), and analysis of their patterns gives the position and the number of double bonds on the acyl groups. Information about the position of each acyl group on the glycerol backbone, however, is not provided by collisionally activated dissociation of [M + NH4]+ ions. On the other hand, ESI- and FAB-produced [M + Na]+ ions fragment to form eight types of ions (named A-J ions) that, like those produced by CRF, are highly structurally informative. The absence of certain series members also carries useful structural information. Interpretation of these patterns enables one to obtain the number of carbons, degrees of unsaturation, and location of double bonds, as well as the positions of acyl groups on the glycerol backbone.     

Electron impact and chemical ionization mass spectrometry of steroidal spirolactones

Electron impact and chemical ionization mass spectra are reported for several steroidal spirolactones and their TMS ethers. The electron impact spectra were characterized generally by low abundance molecular ions and large numbers of fragment ions. Methane chemical ionization spectra exhibited high intensity [M+H]+ and/or [M+H-H2O]+ or [M+H-TMSOH]+ ions with few other fragment ions. Ammonia chemical ionization spectra had intense [M+H]+ and/or [M+NH4]+ ions with a few fragment ions generally formed by loss of H2O or TMSOH from these parent ions. Ammonia chemical ionization gave intense parent ions even for polyhydroxy compounds and their TMS ethers in contrast to methane chemical ionization. The results of this study suggest that a combination of electron impact with ammonia chemical ionization mass spectrometry would offer the best techniques for detection and identification of these compounds in biological fluids.     

The measurement of myocardial tissue gas tensions by mass spectrometry

The electron impact and the hydrogen and methane chemical ionization mass spectra of 5,6-dihydro-2-methyl-1,4-oxathin-3-carboxanilide, the sulfoxide and sulfone derived therefrom, and 2-(2-hydroxyethylthio)-acetoacetanilide enol have been determined. All four compounds show abundant molecular ions in the electron impact spectra and abundant [MH]+ ions in the methane chemical ionization spectra (along with the expected [M + C2H5]+ and [M + C3H5]+ ions), but relatively low [MH]+ ion signals in the hydrogen chemical ionization spectra. From high resolution mass measurements and metastable transitions determined by metastable ion refocusing, electron impact fragmentation mechanisms have been established. For 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide and 5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide the major fragmentation mode involves loss of the anilino radical from [M]+, followed by loss of C2H4. Fragmentation to form the aniline molecular ion increases in importance with increasing oxidation state of the sulfur. In the chemical ionization of these three compounds fragmentation of [MH]+ proceeds in a similar fashion by loss of neutral aniline and by formation of protonated aniline. The electron impact mass spectrum of 2-(2-hydroxyethylthio)acetoacetanilide is dominated by the molecular ion and the aniline molecular ion. However, in the chemical ionization mass spectra characteristic fragment ions involving loss of water and loss of aniline from [MH]+, as well as protonated aniline, are observed and serve to characterize the compound.     

Characteristics of mass fragmentation of steroids by atmospheric pressure chemical ionization-mass spectrometry

The characteristics of the mass spectra of sixty steroids were investigated using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). In APCI-MS, the drift voltage and nebulizer temperature affected the appearance of molecular ions. Solvent adducted ions [M+H+CH3CN]+ and [M+H+H2O]+ were decreased with an increase in drift voltage. The optimum drift voltage differed slightly for each steroid. These sixty steroids were divided into two groups according to their mass spectra profiles: one having a carbonyl group at position 3 together with a double bond at position 4 (group A) and the other bearing a hydroxyl group at the 3 position (group B). In group A, the predominant peak observed corresponded to the protonated molecular ion [M+H]+. The fragment ion corresponding to the elimination of CH2OH, COCH3 and/or COCH2OH from the steroid skeleton appeared as a base peak in some steroids of group A. In group B, predominantly [M+H-H2O]+ and/or [M+H-2H2O]+ ion(s), originating from the loss of water molecules, were observed. Other major ions in this group were the protonated molecular ions. Like other chemical ionization mass spectrometry, the mass spectrum of each steroid was very simple. These results suggest that APCI-MS is a suitable tool for the determination of the mass number of polar, nonvolatile and thermolabile steroids without derivatization.     

Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin-enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal-phase high-performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M + H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in-source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long-chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI-MS/MS via collision-induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB-FA combinations(s) using APCI+(-)MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting approximately 90% of the total ion current in ESI) were composed of at least 25 different LCB-FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB-FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented.     

Structural analysis of xyloglucan oligosaccharides by the post-source decay fragmentation method of MALDI-TOF mass spectrometry: influence of the degree of substitution by branched galactose, xylose, and fucose on the fragment ion intensities

Highly branched xyloglucan oligosaccharides were analyzed by the post-source decay (PSD) fragmentation method of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ratio of [M-Xyl]+ and [M-Gal]+ fragment ion intensities could be used to characterize the degree of Gal substitution at the non-reducing end, because the number of possible chemical species was directly related to their relative ion intensity. The intensity of the [M-Fuc]+ ion was predominantly strong in the fragment spectrum of fucosyl oligosaccharides as the first fragmentation, indicating the fucosyl linkage to be much weaker than the other glycosidic linkages in the MALDI-PSD fragmentation. Setting fragment ion [M-Fuc]+ to the pseudo precursor ion [MF]+, the second fragmentation ions were produced from [MF]+ in the drift region in PSD fragmentation of fucosyl oligosaccharides.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.     

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.     

Acute lung failure induced by tricyclic antidepressants

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC-electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M-H]- ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.     

Structure analysis of trivalent glycoclusters by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

Post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectra were found to be useful for the structural elucidation of a series of tris [2-(glycosylthiourylene)ethyl]amines. The reported fragmentation behaviours of [M + H]+, [M + Na]+ and [M - H]- ions differ from each other significantly; however, they can be compared to tree pruning in every case. Whereas detailed structural information on unprotected glycoclusters is obtained from all PSD experiments, only the positive-ion mode can be used to gain relevant information about the acetylated glycoclusters.     

Tandem mass spectrometry analysis of synthetic opioid peptide analogs

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.     

How potassium affects the activity of the molecular chaperone Hsc70. I. Potassium is required for optimal ATPase activity

Several functions of the 70-kilodalton heat shock cognate protein (Hsc70), such as peptide binding/release and clathrin uncoating, have been shown to require potassium ions. We have examined the effect of monovalent ions on the ATPase activity of Hsc70. The steady-state ATPase activities of Hsc70 and its amino-terminal 44-kDa ATPase fragment are minimal in the absence of K+ and reach a maximum at approximately 0.1 M [K+]. Activation of the ATPase turnover correlates with the ionic radii of monovalent ions; those that are at least 0.3 A smaller (Na+ and Li+) or larger (Cs+) than K+ show negligible activation, whereas ions with radii differing only approximately 0.1 A from that of K+ (NH4+ and Rb+) activate to approximately half the turnover rate observed with K+. Single turnover experiments with Hsc70 demonstrate that ATP hydrolysis is 5-fold slower with Na+ than with K+. The equilibrium binding of ADP or ATP to Hsc70 is unperturbed when K+ is replaced with Na+. These results are consistent with a role for monovalent ions as specific cofactors in the enzymatic hydrolysis of ATP.     

Activities of the enzymes participating in purine and free-radical metabolism in cancerous human colorectal tissues

Liquid chromatography-pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS-MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS-MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 microgram/l or below, whereas detection limits were three-four times higher using MS-MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS-MS. Application of the method for detecting acidic herbicides residues in a "real-world" ground water sample is demonstrated.     

Liquid secondary-ion mass spectrometry of peptides containing multiple tyrosine-O-sulfates

The behavior of peptides containing multiple tyrosine-O-sulfates in liquid secondary-ion mass spectrometry (LSIMS) has been investigated. In the positive-ion spectra of the peptides containing two tyrosine-O-sulfates, Cionin and CCK-associated C-terminal nonapeptide (CAP-9), the completely desulfated [M+H-2SO3]+ ions formed the base peaks, accompanying the significantly less-intense [M+H]+ and [M+H-SO3]+ ions. In the negative-ion spectra of these peptides, the [M-H]- and [M-H-SO3]- ions gave prominent peaks with significantly weaker [M-H-2SO3]- ions. In the case of a peptide containing three tyrosine-O-sulfates, [Tyr(SO3H).1]CAP-9, the completely desulfated [M+H-3SO3]+ ion again formed the base peak in the positive-ion spectrum. On the other hand, the sulfated tyrosine-containing [M+H]+, [M+H-SO3]+, and [M+H-2SO3]+ ions were of negligible abundance compared to the spectra of peptides containing two tyrosine-O-sulfates. We observed an intriguing 'ladder fragmentation pattern' in the negative-ion spectrum of this triply-sulfated peptide. The ladder consisted of the [M-H]-, [M-H-SO3]-, and [M-H-2SO3]- ions, but without the completely desulfated [M-H-3SO3]- ion. These characteristic fragmentation patterns of sulfated tyrosine-containing peptides were considered to bear a close correlation with the inherent acid-lability of a tyrosine-O-sulfate in solution. A possible mechanism has been proposed to explain the fragmentation patterns in the gaseous phase, in which a proton plays a decisive role.     

Glass-reinforced hydroxyapatite: a comprehensive study of the effect of glass composition on the crystallography of the composite

A rapid, simple, and sensitive method is described for the determination of the anomeric configuration of sugar 1-phosphates, sugar nucleotides, and polyisoprenyl-phospho-sugars. Negative-ion electrospray ionization of picomole amounts of glycosyl 1-phosphate derivatives produces an intense signal of the [M-H]-deprotonated molecule which, by collision-induced dissociation, decomposes in a characteristic manner depending on cis/trans configuration of the 2-hydroxyl and phosphate groups of the glycosyl residue. A distinct feature of the product ion spectra of glycosyl 1-P and polyisoprenyl-P-sugars with cis configuration is the presence of abundant ions that correspond to the [M-H2O-H]- dehydration product and the [R-PO4-(C2H3O]- fragment arising from a cleavage across the sugar ring, where R is -H or -polyprenyl/dolichyl for glycosyl 1-P and polyisoprenyl-P-sugar, respectively. These two fragments, [M-H2O-H]- and [R-PO4-(C2H3O)]- are absent from the product ion spectra of sugar 1-P and polyisoprenyl-P-sugars with trans configuration. For sugar nucleotides, compounds with cis configuration produce, in tandem mass spectrometry, only one abundant fragment of nucleoside monophosphate, whereas those with trans configuration give nucleoside diphosphate as a major fragment ion. Accordingly, the anomeric configuration of a glycosyl 1-phosphate derivative can be easily determined by using electrospray-ionization tandem mass spectrometry provided that the glycosyl residue of known absolute configuration has a free 2-hydroxyl group and no other charge location.     

Determination of hepatotoxic pyrrolizidine alkaloids by on-line high performance liquid chromatography mass spectrometry with an electrospray interface

The present study describes the determination of two different types of hepatotoxic pyrrolizidine alkaloids (PAs) and also distinguishing the hepatotoxic PAs from non-toxic ones by both in-source collision-induced dissociation high performance liquid chromatography mass spectrometry (CID-HPLC/MS) and HPLC/MS/MS (CID in the collision cell), using electrospray ionization. The mass spectra provided molecular ions and characteristic fragment ions, which could be used readily for a rapid identification of different types of PAs. Applications of both in-source CID-HPLC/MS and HPLC/MS/MS analytical methods were successful for the determination of PAs in blood samples obtained from rats dosed with PAs and in the PA-containing plant. The results demonstrated that the developed HPLC/MS methods with two different CID techniques provided a very simple and rapid analysis for an unequivocal diagnosis of PA poisoning and for definitive identification of PAs in plants or herbal medicines.     

Multipole storage assisted dissociation, a novel in-source dissociation technique for electrospray ionization generated ions

In this work we present a novel in-source dissociation scheme referred to as multipole storage assisted dissociation (MSAD) for electrospray ionization (ESI) generated ions in which dissociation is effected by employing extended ion accumulation intervals in a high pressure rf-only hexapole assembly prior to mass analysis. Following an extended ion accumulation interval in which ions are confined in the rf-only hexapole, ions are gated out of the hexapole, trapped, and mass analyzed in the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The accumulation region is comprised of an rf-only hexapole ion guide which separates two electrodes, a biased skimmer cone, and an auxiliary 'gate' electrode at the low pressure end of the hexapole. This technique should be applicable to other mass spectrometry platforms compatible with pulsed ionization sources including quadrupole ion traps, and time-of-flight mass analyzers. This concept is demonstrated with the dissociation of a small protein in which selective fragmentation is observed at labile amino acid linkages producing primarily y-type fragment ions.     

Evidence for linkage position determination in cobalt coordinated pentasaccharides using ion trap mass spectrometry

A methodology to determine the linkage position of oligosaccharides is presented. In order to illustrate this technique, several oligosaccharides and disaccharides were ionized by electrospray and analyzed in a Paul trap mass spectrometer. Multiple stage tandem mass spectrometry experiments were used to determine linkage and structural information for the following four cobalt coordinated and singly charged ([M + Co-H]+) pentasaccharides: Lacto-N-fucopentaose I, II, III, and V. In order to differentiate between linkage positions, multiple low energy collision induced experiments with mass selected C type ions have been carried out in an ion trap mass spectrometer. Because of the coordination with cobalt, which directs the dissociation pathways, these C type ions undergo specific fragmentation reactions upon low energy collision induced dissociation. These dissociation pathways are unambiguously dependent on their linkage position, thus allowing differentiation between 1-->2, 1-->3, 1-->4, and 1-->6 linkage positions throughout the oligomers. Studies on various linked disaccharides and N-acetyl-disaccharides, which are smaller constituents of the pentasaccharides, were used to verify and confirm the results obtained from the pentasaccharides.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.