Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry

A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described. Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry. Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed. beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively. These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety. By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety. Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine. Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.     

Improved ternary matrices for the analysis of ferri-pyoverdins by fast atom bombardment mass spectrometry

The use of explosives as matrices, a principle used previously in plasma desorption mass spectrometry, was applied to fast atom bombardment mass spectrometry (FABMS). Ternary matrices were prepared by the addition of picric acid and related compounds to the binary matrix routinely used for the analysis of peptides, viz. thioglycerol-dithiodiethanol. With the new matrices, the spectra of three ferri-pyoverdins (iron-siderophore complexes which are often difficult to analyse by FABMS with standard matrices) showed a significant increase in both intensity and duration of the ion current. They present a valuable tool for the analysis of problematic analytes and for collision-induced dissociation MS/MS experiments.     

alpha-d-glucopyranosyl-, d-alanyl- and l-lysylcardiolipin from gram-positive bacteria: analysis by fast atom bombardment mass spectrometry

Cardiolipin species substituted on O2 of the middle glycerol moiety with alpha-d-glucopyranosyl, d-alanyl and l-lysyl residues were isolated from different gram-positive bacteria. There respective structures were elucidated by positive and negative mode fast atom bombardment mass spectrometry. The structural heterogeneity due to different fatty acid combinations was documented by up to seven molecular ions. General structural features were derived from diagnostic fragment ions, generated by single cleavage at the phosphodiester moieties in both positive and negative ion mode. A diagnostically important fragment ion for d-alanylcardiolipin was observed in the positive ion mode. It arose from double cleavage of the phosphodiester moieties yielding [NaH(Na) PO4-CH2.CH(OCO.CHNH2.CH3) CH2+]+. The fatty acid combinations in the phosphatidyl and diacylglycerol ions make it possible to recognize whether saturated and unsaturated fatty acids were selectively or randomly distributed on the two positions of the glycerol moieties. Molecular structures of cardiolipins, derived from mass spectrometric experiments, are in full agreement with those, elucidated by classical chemical analyses.     

Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.     

Negative ion electrospray ionization tandem mass spectrometry in the structural characterization of penicillins

The mass spectrometry (MS) behaviour of ten commercially available penicillins has been studied by means of electrospray and multiple-stage MS/MS experiments performed using an ion trap instrument. For all the examined compounds negative ions are produced under ESI conditions, with a yield two or three orders of magnitude higher than that observed for positive ions. MSn experiments indicate the occurrence of a fragmentation pathway related to the beta-lactam ring, different from that usually described for positive ions of these compounds, and provide structural information on both the beta-lactam ring and the side chains.     

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.     

Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry

A negative ion electrospray ionization tandem mass spectrometric technique was developed for the analysis of glycerophospholipids. Examination of the product ion mass spectrum of the deprotonated molecular ion provided sufficient information to identify both the class of glycerophospholipid and the molecular weights of the two fatty acid moieties. This technique was applied to the profiling of glycerophospholipids present in the chloroform/methanol extracts of four different bacterial species. The principal bacterial phospholipids detected by this technique were phosphatidylglycerols and diphosphatidylglycerols, accompanied by small amounts of phosphatidylethanolamines for two of the bacterial species examined. The fatty acid composition of the phosphatidylglycerols for each bacteria was determined by tandem mass spectrometry and presented graphically. Differences in the fatty acid composition for each bacterial species were readily apparent from a visual examination of the data sets.     

Characterization of the conformations of antigenic peptides of protein lactate dehydrogenase (LDH-C4) by electrospray ionization mass spectrometry

The conformations of several rationally designed antigenic peptides that mimic, to varying degrees, an antibody-binding region of protein lactate dehydrogenase isozyme (LDH-C4) are investigated by deuterium/hydrogen exchange and electrospray ionization mass spectrometry (ESI-MS). The approach involves monitoring the reverse-exchange of deuterium, incorporated at the labile sites in the peptides, with hydrogen as a function of time by ESI-MS. Idealized forms of a segment of the native antigen are shown to be more conformationally restricted than the native peptide based on level of deuterium that remains incorporated at the labile sites over time. From the number of amide groups of the peptide backbone that retain deuterium, estimates of the helical content of each peptide have been measured that are in close agreement with those determined by Fourier transform infrared (FTIR) spectroscopy in separate experiments. A single amino acid substitution in the idealized helical construct results in a conformational change easily detected by the deuterium exchange ESI-MS method. The approach is shown to be a viable method for characterizing the conformations of protein antigens at the local level and for screening the conformations of antigenic peptides designed to elicit optimal immune responses.     

Analysis of antiretroviral nucleosides by electrospray ionization mass spectrometry and collision induced dissociation

Antiretroviral nucleoside drugs used against the human immunodeficiency virus (HIV) infection have been analyzed using negative ion electrospray ionization (ESI) mass spectrometry and collision-induced dissociation (CID-MS/MS). Mass fragmentation of azidothymidine (AZT), didanosine (ddI), dideoxycytidine (ddC) and dideoxythiacytidine (3TC) were obtained at different cone voltages and collision energies. Fragmentation of purines and pyrimidines occurred by different pathways. For purines (ddI), the fragmentation was similar to those found in endogenous nucleosides; mainly the pseudo molecular ion is present (M-H)- and a cleavage through the glycosidic bond forming (B)- was observed. For pyrimidines (AZT, ddC, 3TC), the fragmentation pathways were different from endogenous nucleosides; for AZT, the fragmentation occurred primarily through the elimination of the azido group in the 3'-position (M-H2-N3)-, whereas ddC and 3TC presented more complex fragmentation patterns. For ddC, fragmentation appeared to be dominated by a retro Diels-Alder mechanism (M-CONH)-. For 3TC, the sulfur atom in the sugar moiety provided greater stability to the charge, producing fragments where the charge resided initially in the dideoxyribose (M-C2O2H6)-.     

Characterization of combinatorial peptide libraries by electrospray ionization Fourier transform mass spectrometry

The advantages of Fourier transform mass spectrometry (FTMS) are precision high mass accuracy measurements and the capability of high resolution, multistage mass spectrometry together with a number of other advanced features. These powerful facilities can be used to rapidly screen complex mixtures without the necessity of chromatographic separations. The example shown here illustrates the use of the high resolving power and accurate mass capabilities of FTMS for the rapid, direct analysis of a complex mixture, which had been ionized by direct infusion electrospray ionization.     

Polysulfated carbohydrates analyzed as ion-paired complexes with basic peptides and proteins using electrospray negative ionization mass spectrometry

Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.     

Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry

The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.     

Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry

The bacitracin antibiotic complex consists principally of bacitracin A, a peptide antibiotic containing seven amino acid residues in a ring and five amino acid residues in a blocked side chain, together with a mixture of minor components presumably related but of unknown structures. A preparative high-performance liquid chromatographic method was developed for isolating the minor components A2, B1 and B2 which were then characterized by amino acid analysis, exact mass fast atom bombardment (FAB) mass spectrometry, FAB tandem mass spectrometry (MS/MS) and electrospray ionization (ESI) mass spectrometry. For bacitracins A (MW 1421), A2 (MW 1421), B1a (MW 1407), B1b (MW 1407), B2 (MW 1407) and F (MW 1419), the side chain sequences were determined by ESI MS/MS and ESI nozzle-skimmer collision-induced dissociation (CID) mass spectrometry and the ring sequences elucidated by ESI nozzle-skimmer CID MS/MS. Relative to bacitracin A, bacitracin A2a has the modified isoleucine residue at position 1 replaced by a modified allo-isoleucine residue, bacitracin B1a has the isoleucine residue at position 8 replaced by a valine residue, bacitracin B1b has the isoleucine residue at position 5 replaced by a valine residue and bacitracin B2 has the modified isoleucine residue at position 1 replaced by a modified valine residue. FAB tandem mass spectra were shown to be consistent with the above structural assignments for the isolated bacitracin components. Structures were also proposed for the trace bacitracin components C1 (MW 1393) and D1 (MW 1379) using ESI MS/MS data obtained from the analysis of the bacitracin complex without isolation.     

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.     

Purification and determination of intact molecular mass by electrospray ionization mass spectrometry of the photosystem II reaction center subunits

A reverse phase high pressure liquid chromatography purification system for the rapid separation of photosystem II reaction center proteins free of salts and detergents is described. This procedure results in the isolation of the three small subunits: alpha- and beta-subunits of cytochrome b559 and PsbI protein, with near base-line resolution between each peak, although the D1 and D2 proteins were partially deconvoluted. The molecular masses obtained by electrospray ionization mass spectrometry for the purified beta-subunit of cytochrome b559, alpha-subunit of cytochrome b559, and the PsbI protein, 4,394.8 +/- 0.4, 9,283.7 +/- 0.8, and 4,209.5 +/- 0.4 Da, respectively, are in excellent agreement with values obtained from previous characterization studies (Sharma, J., Panico, M., Barber, J., and Morris, H. R. (1997) J. Biol. Chem. 272, 3935-3943). Direct electrospray analysis of the D1 and D2 proteins suggests that these components exist in heterogeneous forms. The molecular mass ascribed to a predominant form of the D1 protein, 38, 040.9 +/- 6.5 Da, and the D2 protein, 39,456.1 +/- 7.7, are also in agreement with those expected for the mature nonphosphorylated states of these subunits.     

Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry

A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.     

Analysis of Vero toxins 1 and 2 by high-performance liquid chromatography/electrospray ionization mass spectrometry

PURPOSE: To study the interaction between gabapentin (GBP) and high-protein meals, 12 patients with epilepsy were administered this drug both while in a fasting state and after a high-protein meal. METHODS: After having acquired their informed consent, the patients (suffering from partial complex seizures resistant to other anticonvulsants) were randomly assigned to 2 groups of 6 subjects. Each subject was treated in a fasting state with a single 400 (group A) or 800 (group B) mg GBP oral dose. After 24 h, the GBP dose regimen was repeated, but was given after a high-protein meal. Serum GBP concentrations were measured by LC-Mass at baseline and 0.5, 1, 2, 3, 5, 7, 9, 12, and 24 h. Saliva GBP concentrations were determined at baseline and 2, 4.8, and 12 h. GBP urinary excretion was determined at 0-4, 4-8, and 8-12 h intervals. The following kinetic parameters were calculated: area under the concentration time curve from zero time to 24 h after the dose, AUC 0-24 h; maximal serum concentration, Cmax; time to the maximal serum concentration, Tmax; absorption rate constant, ka; elimination rate constant, beta; elimination half-time, t1/2beta. Student's t test for paired data, with significance assigned at P < 0.05, was used. RESULTS: No statistically significant differences were seen in GBP serum or saliva concentrations or in its urinary excretion (both in A or B group) between fasting and after the high-protein meal. CONCLUSIONS: High-protein meals do not seem to interfere with oral disposition of GBP.     

Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.     

Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry

The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subjected to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and the 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.