ABSTRACT
The oligomeric state and kinetics of ligand binding were measured for wild-type cytoglobin. Cytoglobin has the classical globin fold, with an extension at each extremity of about 20 residues. The extended length of cytoglobin leads to an ambiguous interpretation of its oligomeric state. Although the hydrodynamic diameter corresponds to that of a dimer, it displays a mass of a single subunit, indicating a monomeric form. Thus, rather than displaying a compact globular form, cytoglobin behaves hydrodynamically like a tightly packed globin with a greater flexibility of the N- and C-terminal regions. Cytoglobin displays biphasic kinetics after the photolysis of CO, as a result of competition with an internal protein ligand, the E7 distal histidine. An internal disulfide bond may form which modifies the rate of dissociation of the distal histidine and apparently leads to different cytoglobin conformations, which may affect the observed oxygen affinity by an order of magnitude.
Subject(s)
Disulfides/chemistry , Globins/chemistry , Cytoglobin , Histidine/chemistry , Kinetics , Protein Conformation , Protein Structure, Quaternary , Spectrometry, Mass, Electrospray IonizationABSTRACT
Routine antenatal hemoglobinopathy screening detected a new alpha chain variant that eluted with Hb A(2) on cation exchange high performance liquid chromatography (HPLC) in a lady of Sri Lankan origin who had normal hematological indices. The mutation was identified by electrospray ionization mass spectrometry (ESI-MS) as alpha46(CE4)Phe-->Val, inferring that the variant was due to a single base change at codon 46 (TTC>GTC) of the alpha1- or alpha2-globin genes.
Subject(s)
Hemoglobins, Abnormal/genetics , Point Mutation , alpha-Globins/genetics , Female , Genetic Variation , Humans , Spectrometry, Mass, Electrospray Ionization , Sri Lanka , Young AdultABSTRACT
Population migration has led to the global dispersion of human hemoglobinopathies and has precipitated a need for their identification. An effective mass spectrometry-based procedure involves analysis of the intact alpha- and beta-globin chains to determine their mass, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested chains and low-energy collision induced dissociation of the variant peptide. Using this procedure, a variant was identified as either beta54Val-->Leu or beta54Val-->Ile, since the amino acids leucine and isoleucine cannot be distinguished using low-energy collisions. Here, we describe how hot electron capture dissociation on a Fourier transform-ion cyclotron resonance mass spectrometer was used to distinguish isoleucine from leucine and identify the mutation as beta54(D5)Val-->Ile. This is a novel variant, and we have named it Hb Askew.
Subject(s)
Hemoglobins/chemistry , Isoleucine/chemistry , Leucine/chemistry , Models, Chemical , Spectrometry, Mass, Electrospray Ionization/methods , Spectroscopy, Fourier Transform Infrared/methods , Amino Acid Substitution , Computer Simulation , Electrons , Hot TemperatureABSTRACT
When analyzed by routine cation exchange high performance liquid chromatography (HPLC), the Hb A(1c) peak of a Caucasian diabetic patient from Yorkshire, UK, appeared to be an incompletely resolved doublet. One component (5.5%) eluted at the normal time for Hb A(1c), whereas the other component (6.6%) eluted slightly later. The HPLC trace was otherwise normal. Analysis of the diabetic patient's blood and a tryptic digest thereof by electrospray ionization mass spectrometry (ESI-MS) identified the Hb Belleville trait. To relate Hb A(1c) determined by HPLC to alpha and beta chain glycation determined by ESI-MS, several normal blood samples (5-14% Hb A(1c)) were analyzed by both techniques. The Hb A(1c) levels derived from the alpha and beta chain glycation levels of the diabetic patient (12.9 and 12.4%, respectively) agreed with the sum of the two peaks (12.1%) in the HPLC trace. Similarly, Hb Belleville was detected and identified in another Caucasian, also from Yorkshire, with normal Hb A(1c).
Subject(s)
Chromatography, High Pressure Liquid/methods , Glycated Hemoglobin/analysis , Hemoglobins, Abnormal/analysis , Cation Exchange Resins , Diabetes Mellitus , Humans , Male , Middle Aged , Spectrometry, Mass, Electrospray Ionization , White People , Young AdultABSTRACT
The global dispersion of hemoglobin variants through population migration has precipitated a need for their identification. A particularly effective mass spectrometry (MS)-based procedure involves analysis of the intact globin chains in diluted blood to detect the variant through mass anomalies, followed by location of the variant amino acid residue by direct analysis of the enzymatically digested globins. Here we demonstrate the use of ion mobility separation in combination with this MS procedure to reduce mass spectral complexity. In one example, the doubly charged tryptic peptide from a low abundance variant (4%) occurred at the same m/z value as a singly and a doubly charged interfering ion. In another example, the singly charged tryptic peptide from an alpha-chain variant (26%) occurred at the same m/z value as a doubly charged interfering ion. Ion mobility was used to separate the variant ions from the interfering ions, thus allowing the variant peptides to be observed and sequenced by tandem mass spectrometry.
Subject(s)
Hemoglobins/analysis , Hemoglobins/genetics , Hemoglobin J/analysis , Hemoglobin J/genetics , Hemoglobins, Abnormal/analysis , Hemoglobins, Abnormal/genetics , Humans , Indicators and Reagents , Mass Spectrometry , Spectrometry, Mass, Electrospray IonizationABSTRACT
BACKGROUND: Electrospray ionization mass spectrometry (ESI-MS) can be used for the measurement of glycated adult hemoglobin. Here, we describe the evaluation of ESI-MS for measurement of glycated (GHbF) and acetylated (AcHbF) fetal hemoglobin and the identification by mass of different chains of fetal hemoglobin. METHODS: Blood samples were diluted in an acidic denaturing solvent, desalted with AG 50W-X8 resin and introduced directly into the mass spectrometer. Resulting mass spectra were processed to determine the percentage of GHbF and AcHbF and the gamma-chain masses. RESULTS: The procedure yielded reproducible quantitative assay of GHbF and AcHbF, with coefficients of variation <4.9%. Measurement of alpha-chain glycation was similarly reproducible and is suggested as an alternative marker of glycemic control. Marked increases in glycation occurred in dried spot blood samples, which were related to duration of storage, temperature and glucose concentration. Molecular masses of fetal hemoglobin chains were also determined and in 42 neonates studied, two types A and B were identified, two-thirds were type A with gamma-chain masses corresponding to (G)gamma and (A)gamma. In type B, the relative abundance of the (A)gamma-chain was less and the apparent intensity of the (G)gamma-chain was higher. CONCLUSIONS: ESI-MS can be used for the estimation of GHbF and AcHbF and the accurate measurement of fetal gamma-chain masses. The use of whole blood is preferred for analysis.
Subject(s)
Blood Chemical Analysis/methods , Fetus/metabolism , Glycated Hemoglobin/metabolism , Hemoglobins/metabolism , Acetylation , Adult , Blood Glucose/metabolism , Female , Fetal Blood/metabolism , Glycosylation , Humans , Infant, Newborn , Reproducibility of Results , Sensitivity and Specificity , Specimen Handling , Spectrometry, Mass, Electrospray IonizationABSTRACT
A novel beta chain variant found in combination with beta(0)-thalassemia (thal) was identified in a male infant by electrospray ionization mass spectrometry (ESI-MS). Analysis of the infant's denatured blood and a 30 min. tryptic digest of his blood identified the mutation as beta56(D7)Gly-->Cys, which was confirmed by tandem mass spectrometry (MS/MS). We have named this new variant Hb Leeds. The infant's parents, resident in Yorkshire, UK, but originally from Pakistan, were found to have beta(0)-thalassemia (thal) trait (mother) and Hb Leeds trait (father). Hematological data on the infant's parents and siblings are given. Hb Leeds trait was also found in three unrelated Pakistani adults living in the same area of Yorkshire. Hb Leeds trait in adults appears to have few clinical manifestations, but when combined with beta(0)-thal it led to a more severe anemia in the infant than in the corresponding thalassemic trait in his mother.
Subject(s)
Amino Acid Substitution , Anemia/etiology , Hemoglobins, Abnormal/genetics , beta-Thalassemia/genetics , Anemia/genetics , Family Health , Globins/genetics , Humans , Infant , Male , Pakistan , Spectrometry, Mass, Electrospray Ionization , United Kingdom , beta-Thalassemia/complicationsABSTRACT
Many human hemoglobin variants occur in heterozygotes; that is, the variant and normal hemoglobins are present in the same sample. In a procedure for rapidly identifying such variants by mass spectrometry, mutations that increase the mass by 1 Da require a special approach. One of the steps in this procedure involves digesting the denatured hemoglobin with trypsin and analyzing the resulting peptide mixture by mass spectrometry to identify the mutant peptide. Generally the mutant peptide ion can then be selected as the precursor and sequenced by tandem mass spectrometry to identify or confirm the mutation. However, with heterozygotes in which the mass of the variant is 1 Da higher than normal, the first isotope of the mutant peptide occurs at essentially the same mass as the second isotope of the normal peptide, precluding analysis of the mutant peptide on its own. Product ions from the second isotope of a peptide are doublets, 1 Da apart. The way in which the relative abundance of the components in these doublets varies with the elemental composition of the product ions was predicted from the isotopic abundance of the elements and agreed well with experimental data. These results were applied to the identification of a variant that increases the mass by 1 Da in a heterozygote-that is, beta 131 Gln-->Glu, hemoglobin Camden.
Subject(s)
Glutamic Acid/analysis , Glutamine/analysis , Hemoglobins, Abnormal/chemistry , Mass Spectrometry/methods , Trypsin/metabolism , Humans , Models, ChemicalABSTRACT
The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.
Subject(s)
Pharmaceutical Preparations/chemistry , Electrons , Protons , Spectrometry, Mass, Electrospray Ionization , Tablets , Tandem Mass SpectrometryABSTRACT
Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.
Subject(s)
Cholera Toxin/chemistry , Protein Subunits/chemistry , Models, Molecular , Phase Transition , Protein Structure, Quaternary , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.
Subject(s)
Protein Processing, Post-Translational , Shiga Toxin 1/chemistry , Shiga Toxin 1/metabolism , Hydrogen-Ion Concentration , Phase Transition , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Key to the remarkable ability of vestimentiferan tubeworms to thrive in the harsh conditions of hydrothermal vents are hemoglobins that permit the sequestration and delivery of hydrogen sulfide and oxygen to chemoautotrophic bacteria. Here, we demonstrate that zinc ions, not free cysteine residues, bind sulfide in vestimentiferan hemoglobins. The crystal structure of the C1 hemoglobin from the hydrothermal vent tubeworm Riftia pachyptila has been determined to 3.15 A and revealed the unexpected presence of 12 tightly bound Zn(2+) ions near the threefold axes of this D(3) symmetric hollow sphere. Chelation experiments on R. pachyptila whole-coelomic fluid and purified hemoglobins reveal a role for Zn(2+) ions in sulfide binding. Free cysteine residues, previously proposed as sulfide-binding sites in vestimentiferan hemoglobins, are found buried in surprisingly hydrophobic pockets below the surface of the R. pachyptila C1 molecule, suggesting that access of these residues to environmental sulfide is restricted. Attempts to reduce the sulfide-binding capacities of R. pachyptila hemoglobins by addition of a thiol inhibitor were also unsuccessful. These findings challenge the currently accepted paradigm of annelid hemoglobin evolution and adaptation to reducing environments.
Subject(s)
Hemoglobins/chemistry , Polychaeta/metabolism , Sulfides/metabolism , Zinc/metabolism , Animals , Base Sequence , Binding Sites , Crystallization , Hydrophobic and Hydrophilic Interactions , Molecular Sequence DataABSTRACT
Within a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry. Hemoglobin Lepore-Boston-Washington occurs mainly in heterozygotes, where it comprises approximately 10% of the total non-alpha-chains, the dominant non-alpha-chain being the normal beta (approximately 90%). Furthermore, Hemoglobin Lepore-Boston-Washington has an average molecular mass (15,865.23 Da) that is only 2 Da lower than that of the normal beta-chain (15,867.24 Da). Consequently, it cannot be resolved from the normal beta-chain by mass spectrometry. Here we show how Hemoglobin Lepore-Boston-Washington was identified directly from the diluted blood of a heterozygote by analyzing the product ions from the Lepore-Boston-Washington and normal beta-chain ions without prior separation of the individual chains. This study shows the potential of the tandem mass spectrometry for identifying a minor component in an unseparated mixture of proteins.
Subject(s)
Hemoglobins, Abnormal/analysis , Hemoglobins, Abnormal/genetics , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Electrospray Ionization/standards , Amino Acid Sequence , Heterozygote , Humans , Molecular Sequence Data , Reproducibility of ResultsABSTRACT
Neuroglobin displays a hexacoordination His-Fe-His in the absence of external ligands such as oxygen. The observed oxygen affinity therefore depends on the binding rates of both oxygen and the competing distal histidine. Furthermore, the binding properties depend on the presence of an internal disulfide bond. In the case of human neuroglobin, cysteines at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. For cytoglobin, the cysteine residues at positions A7 and GH4 may also form a disulfide bond. Mass spectrometry, ligand binding, and thiol accessibility studies were used to study the role influence of these disulfide bonds. Mutation of specific cysteines, or reduction to break the S-S bond, led to a large decrease in the observed oxygen affinity of human neuroglobin, mainly due to a decrease in the histidine dissociation rate. This suggests a novel mechanism for the oxygen binding; reduction of the disulfide bond would provoke the release of oxygen.
Subject(s)
Disulfides/chemistry , Globins/chemistry , Heme/chemistry , Nerve Tissue Proteins/chemistry , Cysteine/chemistry , Humans , Mass Spectrometry , Neuroglobin , Oxygen/metabolismABSTRACT
The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains (M), a1 = 16535 Da, a2 = 17171 Da and a3 = 17315 Da, five nonglobin linker chains, L1 = 24512 Da, L2 = 24586 Da, L3 = 24979 Da, L4 = 25006 Da, and L5 = 25566 Da and two subunits of 32950 Da and 33125 Da. ESI-MS of the denatured, reduced Hb showed that the latter were disulfide-bonded heterodimers (D) of globin chains b1 = 16322 Da and b2 = 16499 Da with chain c = 16632 Da. Time-of-flight ESI-MS of the Hb at pH 3.8, 4.5, 5.0, 5.8 and 7.0 revealed a distribution of charge states from 32(+) to 37(+) with masses decreasing from 211 to 208.5 kDa with increase in cone voltage from 60 to 160 V, indicating the presence of a subassembly comprising 12 globin chains. The subunit composition 6M + 3D + 12h, where M = 16993 Da and D = 33004 Da are the weighted masses and h = 616.5 Da, provides a calculated mass, 208.37 kDa that is closest to 208.5 kDa. Our experimental findings are consistent with the bracelet model of HBL Hbs, verified by the recent low-resolution crystal structure of Lumbricus Hb, wherein an HBL arrangement of 12 globin dodecamer subassemblies is tethered to a central complex of 36 linker chains for a total mass of 208.37 x 12 + 24.94 x 36 = 3398 kDa.
Subject(s)
Hemoglobins/chemistry , Leeches/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Molecular Weight , Protein DenaturationABSTRACT
Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.
Subject(s)
Globins/metabolism , Nerve Tissue Proteins/metabolism , Oxygen/metabolism , Cysteine/chemistry , Cytoglobin , Disulfides , Globins/chemistry , Histidine/chemistry , Humans , Kinetics , Ligands , Models, Molecular , Nerve Tissue Proteins/chemistry , Neuroglobin , Oxidation-Reduction , Protein Binding , Recombinant Proteins , Spectrometry, Mass, Electrospray IonizationABSTRACT
Mass spectrometry has a basic limitation when human hemoglobin variants are analyzed, because it cannot resolve two globin chains that differ in mass by <6 Da. Several common beta-chain variants differ by 1 Da from normal and, hence, when present in heterozygotes, are not resolved from the normal beta-chain. Normal and variant chains appear together in the spectrum as a single entity, whose mass is the abundance weighted mean of the two chains. Here we show that such heterozygotes can be detected in 500-fold diluted blood by accurately measuring the mass of the beta-chain using an electrospray ionization quadrupole instrument and the alpha-chain for internal mass calibration. A statistical analysis of the normal beta-chain mass (n = 86) showed that the standard deviation (SD) of the mean was <+/-0.05 Da (<+/-3.2 ppm). Hence, at the 95% confidence level (+/-2 SD), an abnormal alpha- or beta-chain differing by 1 Da from normal should be detectable in a heterozygote provided its abundance is >10% of total alpha- or beta-chains, respectively. Variants whose masses lay between 1 and 4 Da from normal were detected in 19 heterozygotes. Moreover, the proportion of each variant estimated from the mass change correlated with the proportion determined by cation-exchange HPLC. Variants were assigned to the alpha- or beta-chain by combining the sign of the mass change with the polarity change inferred from electrophoretic data. This procedure could be used for screening clinically significant hemoglobin variants.
Subject(s)
Hemoglobins, Abnormal/chemistry , Hemoglobins, Abnormal/genetics , Calibration , Electrophoresis , Heterozygote , Humans , Mutation/genetics , Reproducibility of Results , Spectrometry, Mass, Electrospray IonizationABSTRACT
We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.