Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.     

Protein analysis by electrospray ionization mass spectrometry

The applicability of electrospray ionization (ESI) mass spectrometry to protein analyses has been studied. The molecular weight of hen egg lysozyme (HEL) was determined with an accuracy of +/- 2 u. The choice of solvents and additives in sample preparations was important to achieve high sensitivity as well as high precision of molecular weight measurements.     

Direct monitoring of sequential enzymatic hydrolysis of peptides by thermospray mass spectrometry

A procedure for peptide sequencing using an immobilized exopeptidase column directly coupled to a thermospray mass spectrometer is described. The amino acids sequentially released from the C-terminus of the peptide chain are directly introduced into a thermospray ion source by a flowing aqueous buffer. The buffer is essential for the direct production of ions from solution. The method eliminates the need to derivatize the amino acids for detection and, by comparison to standard injections, amino acid sequence information can be obtained in less than two minutes. With the present configuration, detection limits are typically in the low picomolar range.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Quantifying carbohydrate-protein interactions by electrospray ionization mass spectrometry analysis

The development of analytical methods capable of characterizing carbohydrate-protein interactions, which are critical for many biological processes, represents an active area of research. Recently, the direct electrospray ionization mass spectrometry (ESI-MS) assay has emerged as a valuable tool for identifying and quantifying carbohydrate-protein complexes in vitro. The assay boasts a number of strengths, including its simplicity, speed, low level of sample consumption, and the unique ability to directly probe binding stoichiometry and to measure multiple binding equilibria simultaneously. Here, we describe the implementation of the direct ESI-MS assay for the determination of carbohydrate-protein binding stoichiometries and affinities. Common sources of error encountered with direct ESI-MS analysis of carbohydrate-protein interactions are identified along with strategies for minimizing their effects. The application of ESI-MS and a catch-and-release strategy for carbohydrate library screening are also described. The utility of the direct ESI-MS assay can be extended by combining the technique with competitive protein or ligand binding. An overview of these "indirect" ESI-MS methods is given, as well as examples of recent applications.     

Real-time monitoring of enzymatic hydrolysis of galactomannans

Enzymatic hydrolysis was monitored in real-time using time dependent static light scattering (TDSLS) for a variety of galactomannans from native Brazilian flora. alpha-Galactosidase, which strips only the (1-6)alpha-D galactose side groups, and beta-mannanase, which hydrolyses only the (1-4)beta-D mannan main chain into oligosaccharides were investigated separately and in combination. The time-dependent signatures matched those describing side-chain stripping for galactosidase, whereas those resulting from the action of mannanase followed the signature typical of random backbone cleavage. Use of both enzymes together required that the TDSLS theory of polymer degradation be extended to the case where random backbone cleavage sites appear as side chains are stripped by the first enzyme. Whereas galactosidase allowed mannanase to access more backbone cleavage sites as time passes, leading to a higher degree of hydrolysis, there was no increase in rate constants. The distribution of random fragments in the case of mannanase digestion alone followed reasonably well the predictions for random cleavage of a single-strand polymer with a restricted number of cleavage sites. The fragment distributions were evaluated by size exclusion chromatography.     

Ambient molecular imaging by desorption electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sample preparation other than production of microtome tissue slices and does not involve the use of ionization matrices. This greatly simplifies the procedure and prevents the redistribution of analytes during matrix deposition. Images are obtained by continuously moving the sample relative to the DESI sprayer and the inlet of the mass spectrometer. The timing of the protocol depends on the size of the surface to be analyzed and on the desired resolution. Analysis of organ tissue slices at 250 microm resolution typically takes between 30 min and 2 h.     

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.     

Lipid profiling of lipoproteins by electrospray ionization tandem mass spectrometry

Lipoproteins are of fundamental importance for the lipid transport and cardiovascular disease. The function and metabolism of lipoproteins is intimately linked to the biophysical properties of their surface lipids. Although a number of disease associations were found for lipid species in plasma, only a few studies reported lipid profiles of lipoproteins. Here, we provide an overview of techniques for lipoprotein separation, methods for lipid species analysis based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) as well as data from recent lipidomic studies on lipoprotein fractions. We also discuss the different analytical strategies and how lipid profiling can expand our understanding of the biology and structures of lipoproteins.     

Molecular weight determination of plasmid DNA using electrospray ionization mass spectrometry.

Ionization and molecular weight (MW) determination of megadalton size plasmid DNA has been achieved using electrospray ionization (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. DNA molecules were shown to remain intact through electrospray ionization by collection on a specially prepared surface, followed by agarose gel electrophoresis. Individual highly charged ions of plasmid DNA produced by ESI were trapped in an FTICR cell for up to several hours and reacted with acetic acid to induce charge state shifts. Measurements of mass-to-charge ratios for these multiple peaks arising from charge state shifting give MW measurements of individual ions with an average accuracy of 0.2%. The MW distribution was obtained by measurements for a number of individual ions from the same sample [plasmid DNA: pGEM-5S MW(cal) = 1.946 MDa], yielding a MW(obs) of 1.95 +/- 0.07 MDa for ions clustered in the vicinity of the expected MW.     

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.     

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.     

Detection of a novel variant human hemoglobin by electrospray ionization mass spectrometry

A novel hemoglobin variant was detected by electrospray ionization mass spectrometry. Hb Zurich-Hottingen is characterized by an Asn --> Ser replacement in the alpha-chain at position 9 as confirmed by DNA analysis. This hemoglobin variant is silent in isoelectric focusing, reversed-phase chromatography, and cation-exchange chromatography. The mutant alpha-chain was detectable only with electrospray mass spectrometry by its mass shift of -27 Da. The carrier was found to be heterozygous for the new hemoglobin variant. These results illustrate the power of ESI mass spectrometry for hemoglobin analysis.     

Study on noncovalent complexes of cytotoxic protoberberine alkaloids with double-stranded DNA by using electrospray ionization mass spectrometry

The noncovalent complexes of four cytotoxic protoberberine alkaloids that is, berberine, palmatine, jatrorrhizine, and coptisine with double-stranded oligodeoxynucleotides d(AAGAATTCTT)(2) were investigated by electrospray ionization mass spectrometry. These four active components from Chinese herbal medicines showed both 1:1 and 1:2 binding stoichiometries, independent on the alkaloid-to-DNA ratios. Binding affinities in the order of palmatine> or =jatrorrhizine>coptisine>berberine with d(AAGAATTCTT)(2) were obtained. Additionally, the preliminary results indicated that berberine had some sequence selectivities.     

Determination of branched beta-cyclodextrin-prostaglandin complexes using electrospray ionization mass spectrometry

Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of beta-cyclodextrin (betaCD) or branched betaCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-betaCD)-prostaglandins (PGs: PGA(2), PGD(2), PGE(1), PGE(2), PGF(2alpha) and PGJ(2)) complexes, i.e., betaCD-PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched betaCD-PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a betaCD-PGE(2) complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of betaCD-PGE(2) (Prostarmon E tablet).     

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.     

Analysis of fluorescently labeled oligosaccharides by capillary electrophoresis and electrospray ionization mass spectrometry

Neutral oligosaccharides were fluorescently conjugated with 7-amino-1, 3-naphthalenedisulfonic acid. A mixture of fluorescently labeled chitobiose, chitotriose, and chitotetrose were successfully separated by preparative capillary electrophoresis (CE) and the individual components characterized by electrospray ionization-mass spectrometry (ESI-MS). By combining fluorescent labeling with CE, the use of highly specific exoglycosidases and ESI-MS, a more structurally complex N-linked glycan was analyzed.     

Measuring dissociation constants of RNA and aminoglycoside antibiotics by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to determine the dissociation constants (K(D)s) and binding stoichiometry for tobramycin and paromomycin with a 27-nucleotide RNA construct representing the A-site of the 16S ribosomal RNA. K(D) values determined by holding the ligand concentration fixed are compared with K(D) values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt present in the solution on the K(D) measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS is holding the RNA target concentration fixed below the expected K(D) and titrating the ligand. K(D) measurements should also be carried out at as high as possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding sites were found with K(D1) = 352 nM and K(D2) = 9 microM. For paromomycin, there is only one binding site with K(D) = 52 nM.     

General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry

An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors. The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems. This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques. More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates. This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5'-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5'-triphosphate and ATP substrates.     

Calcium and zinc complexes of pyrroglutamate analogs detected by electrospray ionization mass spectrometry

The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI–MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L₂M and LM species. All tested compounds are better complexing agents for calcium than for zinc.