Direct Molecular Weight Determination Research Articles

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument.

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

Computational analysis of the accuracy in the evaluation of the mean molecular weight of petroleum oils from the ASTM Standard D 2502-92

It is well known that the kinematic viscosity of a petroleum oil is very sensible to small changes in temperature. In this work the author takes into account the experimental errors involved in the measurements of the kinematic viscosity of petroleum oils and applies the error theory to a set of equations recently published that successfully fit the data provided by the ASTM standard D 2502–92. This standard proposes a method for the evaluation of the mean molecular weight (relative molecular mass) of petroleum oils from kinematic viscosity measurements and uses a viscosity-molecular weight chart, which unfortunately involves unavoidable interpolation errors. Due to the relative complexity of the new set of equations, a PC program is designed and used in order to obtain some instructive figures that show the behaviour of the relative error linked to the mean molecular weight of petroleum oils versus different parameters. All the figures are plotted by supposing a relative error in the kinematic viscosity measurements of 1%. Our results show that, under these conditions, the relative errors associated to the evaluation of the mean molecular weight of petroleum oils are always below 2.6%, which permit us to recommend this computational method as a useful and quick alternative to the classic techniques for direct molecular weight determination.

“Unknown Genome” Proteomics: A New NAD(P)-dependent Epimerase/Dehydratase Revealed by N-terminal Sequencing, Inverted PCR, and High Resolution Mass Spectrometry

We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

Direct molecular weight determination in the evaluation of dissolution methods for unreduced glutenin

A comparison of two dissolution methods, gentle stirring and sonication, for solubilising the ultra-large wheat protein glutenin was made based on a direct determination of the molecular weight of dissolved glutenin. The possible physical breakdown of glutenin by sonication was evaluated. Glutenin was isolated by extraction with hydrochloric acid and freeze dried. The extracted glutenin was dissolved either by gentle stirring at 4 °C in phosphate buffer pH 6.8 containing sodium dodecyl sulphate (SDS) or by ultrasonication in the same SDS containing buffer immersed in an ice-bath. The weight average molecular weight and the molecular weight distribution of the dissolved material were determined using asymmetrical flow field-flow factionation using a multiangle light scattering detector coupled on-line with a refractive index detector. These determinations showed that sonication caused physical breakdown of the ultra-large glutenin proteins. The average molecular weight was found to be 5×106 after sonication and 2×107 after gentle stirring.

Identification of fluorescein-5'-isothiocyanate-modification sites in proteins by electrospray-ionization mass spectrometry.

Model peptides and proteins, such as hen eggwhite lysozyme, have been modified with fluorescein-5'-isothiocyanate (FITC) to yield the corresponding fluorescein-thiocarbamoyl (FTC) conjugates (N, N'-disubstituted thiourea and dithiourethane adducts). The extent of FITC incorporation, i.e., number of modified residues, has been identified by direct molecular weight determination using matrix-assisted laser desorption-ionization and electrospray-ionization mass spectrometry (MALDI-MS; ESI-MS). A specific fragmentation by cleavage of the FTC moiety from modified residues occurs by nozzle-skimmer dissociation in ESI mass spectra at increased declustering potential. This fragmentation pathway is easily obtained and renders ESI-MS an efficient tool for the characterization of FITC-modified proteins, and identification of modification sites in FTC-peptide mixtures.

A mass spectrometric approach to the characterization of protein folding reactions

The arsonous acids melarsen oxide and pyridinyl-3-arsonous acid were successfully applied for chemical trapping of otherwise tran sient (un)folding intermediates. The trapped and arsonous acid-modified peptides and proteins were amenable to direct molecular weight determination by mass spectrometry, e.g. by nanoelectrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization mass spectrometry. Arsonous acids exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups providing detailed tertiary structure information, (ii) high solubility as monomeric and oligomeric deriva tives in aqueous solution, (iii) adding a relatively large mass increment to proteins upon single modification and (iv) performing fast and specific modification of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of arsonous acids. Bis-cysteine thiol-selective modification of vasopressin with pyridinyl-3-arsonous acid was confirmed by Fourier transform ion cyclotron resonance mass spectrometry measurements. The major product obtained by denaturing unfolding of bovine pancreatic trypsin inhibitor (BPTI) and trapping with melarsen oxide eluted like folded BPTI in high-performance liquid chromatography analyses, indicating a “native-like” structure of this folding intermediate. The results obtained from the investigated model compounds suggest a general applicability of arsonous acids for selective bis-thiol modifications, enabling the study of protein folding reactions by mass spectrometry.

Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocarbamoyl-sulfoxide

Disulfiram (DSF) is presently the only available drug used in the aversion therapy of recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase (ALDH), leading to high blood levels of acetaldehyde. The in vitro inhibition of ALDH by DSF and its metabolites was systematically studied by combined enzyme inhibition assay with direct molecular weight determination of the same sample using electrospray ionizationmass spectrometry (ESI-MS). Enzyme activity was measured after incubating yeast ALDH (yALDH) with excess concentrations of DSF, methyl diethyldithiocarbamate (MeDDC) and methyl diethylthiocarbamoyl-sulfoxide (MeDTC-SO) and then subjected to analysis by ESI-MS. Addition of DSF resulted in complete enzyme inhibition; however, ESI-MS analysis demonstrated no discernible shift in molecular weight, indicating that no intermolecular adduct was formed with the protein. Treatment of yALDH with MeDTC-SO also completely abolished yALDH activity with a concomitant increase of +≈100 Da in the molecular mass of the enzyme. This indicated formation of a covalent carbamoyl protein adduct. Furthermore, the effects of dithiothreitol (DTT) were examined on samples of inhibited protein in vitro. At pH 7.5, DTT completely reversed inhibition after DSF treatment. yALDH inhibited by MeDTC-SO could not be recovered by DTT at pH 7.5, but at pH 9 the enzymic activity was fully restored and a mass loss of-100 Da was noted. This observations are consistent with mechanisms where inhibition of yALDH by DSF in vitro involves oxidation of the active site, whereas MeDTCSO forms a covalent adduct with the protein in vitro resulting in cessation of enzyme activity.

MALDI‐TOF mass spectrometry in polymer analytics, 2. Molecular weight analysis of rigid‐rod polymers

AbstractMolecular weight analysis of rigid‐rod polymers by gel permeation chromatography (GPC) is known to give overestimated values if polystyrene is used for calibration. In this work matrix‐assisted laser desorption/ionisation time of flight (MALDI‐TOF) mass spectrometry was applied for the first time toward direct molecular weight determination of a rigid‐rod type tetrahydropyrene (THP) polymer. The obtained results were supported by investigation of a homologous series of GPC‐separated oligomers with the respective structure. As opposed to common MALDI‐TOF mass spectra, monodisperse THP oligomers reveal two unusual facts: (i) Beside the formation of molecular ions fragmentation also takes place. (ii) The radical ions are detected instead of cationised species. The fully characterised THP oligomers were also used to calibrate the GPC measurements of the THP polymers. The number‐average molecular weight (M̄n), weight‐average molecular weight (M̄w) and polydispersity (M̄w/M̄n) data obtained from the oligomer‐calibrated GPC compare well to those values measured by MALDI‐TOF mass spectrometry.

A new approach to characterising carboxymethylcelluloses by size exclusion chromatography

Sodium carboxymethylcelluloses are analysed by size exclusion chromatography using three detectors online. First, the molecular weight distribution was established using either direct molecular weight determination or the universal calibration procedure. The agreement between the two series of data is good and seems to indicate that the K and a parameters of the Mark-Houwink relationship are not strongly affected by the degree of substitution (DS) at least in the range tested. In addition, it was demonstrated that dn dc is independent of DS. The amount of salt rejected depends on the quantity of polymer injected and on its charge densities. A quantitative analysis of salt rejection was performed for three samples. The charge distribution was superimposed with the molecular weight distribution. The conductimetric detector was shown to be valuable for describing charge distributions only for low charge parameter ( λ < 1). For the three samples tested we showed that the charge density does not change with molecular weight as reflected by the constancy of the ratio between the conductimetric and refractometric signals.

Direct molecular weight determination of resin-bound oligopeptides using252Cf plasma-desorption mass spectrometry

Plasma-desorption mass spectrometry is proved to be capable of the direct molecular weight determination of totally protected resin-bound oligopeptides. The molecular weight measured is the molecular weight of the fully protected oligopeptide including the molecular weight of the linker, which connects the oligopeptide and the polystyrene resin. This method makes it possible to check the success of (a step in) the synthesis of a peptide before its deprotection or cleaving from the resin. This can make it a strong tool in the analysis of resin-bound oligopeptides.

A Multiple Variable-Angle Light Scattering Detector for Gel Permeation Chromatography.

A light scattering detector has been designed and constructed for use with gel permeation chromatographs. The detector measures light scattered from the eluting sample simultaneously at nine scattering angles, and is connected to a computer-controlled display which exhibits the angular dependence of the scattering in real time while the sample is eluting. Use of the light-scattering detector in conjunction with the usual concentration-sensitive detector allows the direct determination of molecular weight as a function of elution volume, thereby making the chromatographic system “self-calibrating.” Tests of the system with a series of linear and branched polystyrenes suggest that it will be a useful tool for the study and characterization of branched polymers.

Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectrometry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.

Mass Spectrometric Peptide Mapping Analysis and Structural Characterization of Dihydrodiol Dehydrogenase Isoenzymes

The direct molecular weight determination and structural analysis of polypeptides and peptide mixtures have become amenable by the recent development of fast atom bombardment (FABMS) and 252Cf-plasma desorption (PDMS) mass spectrometry. FABMS and PDMS peptide mapping, i.e., the direct analysis of peptide mixtures resulting from proteolytic digestion, have been developed as powerful methods for the structural characterization of epoxide-metabolizing isoenzymes. The major advantage of this approach is provided by the selectivity of the endoproteolytic cleavage, combined with the specific and accurate molecular weight determination of complex digest mixtures containing peptides up to several thousands daltons in size. Furthermore, the mass spectrometric peptide mapping analysis can be combined with a range of protein-chemical modification reactions and with sequential degradation such as by carboxypeptidases. Both FABMS and PDMS peptide mapping have already been successfully applied to the structural differentiation of glutathione transferase and epoxide hydrolase isoenzymes in cases where references sequence data for at least one isoenzyme form was available. In the application described here, for a series of dihydrodiol dehydrogenase (DDH) isoenzymes with hitherto undetermined primary structures, a direct correlation between the structural differentiation from peptide mapping data and differences in their substrate specificities could be demonstrated. The mass spectrometric peptide mapping analysis of isoenzymes proved to be an efficient basis for the elucidation of the structure of one major DDH isoenzyme form; partial sequence data for this protein are reported.

Mass spectrometric peptide mapping analysis and structural characterization of dihydrodiol dehydrogenase isoenzymes.

The direct molecular weight determination and structural analysis of polypeptides and peptide mixtures have become amenable by the recent development of fast atom bombardment (FABMS) and 252Cf-plasma desorption (PDMS) mass spectrometry. FABMS and PDMS peptide mapping, i.e., the direct analysis of peptide mixtures resulting from proteolytic digestion, have been developed as powerful methods for the structural characterization of epoxide-metabolizing isoenzymes. The major advantage of this approach is provided by the selectivity of the endoproteolytic cleavage, combined with the specific and accurate molecular weight determination of complex digest mixtures containing peptides up to several thousands daltons in size. Furthermore, the mass spectrometric peptide mapping analysis can be combined with a range of protein-chemical modification reactions and with sequential degradation such as by carboxypeptidases. Both FABMS and PDMS peptide mapping have already been successfully applied to the structural differentiation of glutathione transferase and epoxide hydrolase isoenzymes in cases where references sequence data for at least one isoenzyme form was available. In the application described here, for a series of dihydrodiol dehydrogenase (DDH) isoenzymes with hitherto undetermined primary structures, a direct correlation between the structural differentiation from peptide mapping data and differences in their substrate specificities could be demonstrated. The mass spectrometric peptide mapping analysis of isoenzymes proved to be an efficient basis for the elucidation of the structure of one major DDH isoenzyme form; partial sequence data for this protein are reported.

Direct Determination of Molecular Weight and Its Distribution by the Absolute Calibration Method Using the On-Line GPC/NMR

The gel permeation chromatography of isotactic PMMAs containing exactly one t-butyl group per chain at the chain end was performed on a chromatograph linked to a 500 MHZ 1H NMR spectrometer. The M̅n of the solute PMMA can be directly determined from the intensities of 1H NMR signals due to t-butyl and methoxy groups with a time resolution of 24 s. Tapered structures at the ends of the NMR flow-cell are effective for preventing broadening of the elution band. Log (M̅n) vs. elution time plots for the three PMMA samples (M̅n=3160, 5260, 12600) fall on a single straight line. The results demonstrate the feasibility of the on-line GPC/NMR as an absolute calibration method for GPC.

ABSOLUTE MOLECULAR WEIGHT DISTRIBUTION OF NITROCELLULOSE

Abstract Being one of the oldest known polymers, nitrocellulose has been the subject of study for over a century. It was initially used solely for military purposes; however, since the end of World War I its usage has expanded into the fiber and coatings industries where it has been a mainstay to the present day [1, 2]. General interest in the molecular weight characterization of nitrocellulose may be classified into two categories. The firstand more obvious reason for the relevance of this parameter is that the molecular weight and molecular weight distribution profoundly influence the properties of the product and affect the processability of the material, For instance, the molecular weight (hence, the viscosity of this material) is one of the most important factors that needs to be considered in the determination of the nonvolatile content in lacquers [21]. The second reason is that it is a soluble derivative of cellulose, for which direct molecular weight determination is problematic due to solubility...

Direct and automatic capillary GC analysis for molecular weight determination and distribution in crude oils and condensates up to C20

AbstractA direct and automatic method has been developed for molecular weight (MW) determination and distribution in crude oils and condensates up to C20 (340°C) by gas chromatography. Chromatographic separation has been achieved with a 50 m OV‐1 fused silica capillary column, fitted with a pre‐column to eliminate the fraction boiling above 340°C. The internal standard is added for quantification. Approximately 550 peaks have been identified by the use of GC/MS on different paraffinic, naphthenic, and aromatic crude oils or condensate samples. From identification and by correlation with MS group‐type analysis, a carbon number and a hydrocarbon group are attributed to each peak or group of peaks. Further molecular weights have been calculated from these groups. A comparison between results from this GC method and cryometry of narrow and wide fractions shows good agreement, within the precision of cryometry. An automatic identification by computer using programmed temperature indices was used for the direct determination of molecular weight. The final report gives weight percentage, molar percentage, and molecular weight for every boiling range group and hydrocarbon group.

Determination of the molecular weight of detergent-solubilized enzymes by sedimentation equilibrium in an air-driven ultracentrifuge

Detergent solubilization of γ-glutamyl transpeptidase was deduced from measuring sedimentation equilibrium behaviour in an air-driven ultra-centrifuge. Sequential fractionation of the tube contents and analysis of the radial concentration dependence permits direct determination of molecular weight. Studies using D 2O and H 2O indicate that the Triton X-100 solubilized enzyme has a molecular weight of approximately 175,000 and contains 55% bound detergent. The glycoprotein nature of the enzyme is demonstrated by its interaction with concanavalin A.

Ammonia gas: an improved reagent for chemical ionization mass spectrometry of bile acid methyl ester acetates

The ammonia chemical ionization mass spectra of 28 methyl ester acetate derivatives of bile acids and related compounds have been determined by gas-liquid chromatography-mass spectrometry. Advantages of ammonia ionization over the previously studied isobutane ionization include a 130-270% enhancement in the sensitivity of base peak monitoring, and direct determination of molecular weight from the base peak (M + NH4+) in the mass spectrum of any of the derivatives. Minor ions in the ammonia spectra also allow selective detection of 3-keto compounds and can indicate unsaturation or double bond conjugation in the molecule. The significance of these studies for the detection and quantitation of bile acids is discussed.

Structure determination of flavonoid disaccharides by mass spectrometry

The mass spectra of 13 perdeuteriomethylated flavonoid disaccharides comprising flavones, flavanones, flavonols and an isoflavone are reported. It is shown that their fragmentation pattern allows unequivocal differentiation according to the structural type of the aglycone, the sugar sequence and the position of the interglycosidic linkage. Moreover, information regarding substitution on the aglycone can be obtained. All compounds except one exhibited molecular ion peaks thus allowing direct molecular weight determination.