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Mass Determination (mass + determination)
Kinds of Mass Determination Selected AbstractsIn-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2002S. Kilz Abstract Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide brigded, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF,pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O -glycosylated proteins up to 150 kDa after SDS-PAGE separation. Copyright © 2002 John Wiley & Sons, Ltd. [source] Characterization of glyco isoforms in plasmaderived human antithrombin by on-line capillary zone electrophoresis-electrospray ionization-quadrupole ion trap-mass spectrometry of the intact glycoproteinsELECTROPHORESIS, Issue 13 2004Uwe M. Demelbauer Abstract The carbohydrate structures of five isoforms of ,-AT and two isoforms of ,-AT were determined by applying capillary zone electrophoresis (CZE) on-line coupled to electrospray ionization-mass spectrometry (ESI-MS) using an ion-trap analyzer. For the AT preparations gained from a plasma pool at least semiquantitative information on the isoform-distributions could be gained. Unlike to the commonly used approaches starting from enzymatically treated glycoproteins, this approach deals with intact proteins. The high accuracy of the molecular mass determination obtained by the ion-trap analyzer allows one to calculate and ascertain the carbohydrate composition assuming no variations in the protein moiety of AT and to exclude or confirm the presence of the potential post-translational or other modifications. Therefore, the direct coupling of CZE with ESI-MS does not only represent a fast alternative technique to two-dimensional electrophoresis (2-DE) but serves as a method which provides structural information complementary to that gained from peptide mapping methods. [source] Pigments and proteins in green bacterial chlorosomes studied by matrix-assisted laser desorption ionization mass spectrometryFEBS JOURNAL, Issue 2 2000Søren Persson We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 µL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB. [source] Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibrationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2002Omar Belgacem Abstract Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de- N -glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of ±0.12 and ±0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip® technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III, and AT-III,, were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de- N -glycosylation (by means of PNGase F) of the ,- and ,-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (,0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N - and O -glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous ,-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N -glycans. Copyright © 2002 John Wiley & Sons, Ltd. [source] Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus speciesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001Gerold Bacher Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source] Straightforward Determination of the Degree of N -Acetylation of Chitosan by Means of First-Derivative UV SpectrophotometryMACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 14 2008Ricardo M. P. da Silva Abstract First-derivative UV spectrophotometry is shown to be a reliable method for the determination of the degree of N- acetylation of chitosan samples. A mathematical expression is derived that allows to determine the DA directly from the mass concentration of a chitosan solution and the first derivative of its UV spectrum at 202 nm, thus eliminating the need for empiric correction curves for highly deacetylated samples. A procedure is proposed for the accurate mass determination of the hygroscopic chitosan. The proposed approach facilitates the routine determination of the DA, especially when using potent multiwell microplate readers, which allow hundreds of samples to be measured in just a few minutes. [source] A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Ricarda Niggeweg Abstract MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode , implemented on a quadrupole-TOF instrument , that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain. [source] An electrospray mass spectrometric method for accurate mass determination of highly acid-sensitive phosphoramiditesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2008Zoltán Kupihár An accurate mass determination method utilizing electrospray ionization mass spectrometry is described for analysis of several different types of phosphoramidites that are extremely acid-sensitive compounds. An earlier method, which applied a LiCl/acetonitrile system, was extended for this special application by using polymeric standards including poly(ethylene glycol) (PEG), poly(ethylene glycol) dimethyl ether (PDE) and poly(propylene glycol) (PPG). Concentrations of standards, samples and LiCl were optimized and potential impurities that affect the analyses were also investigated. Copyright © 2008 John Wiley & Sons, Ltd. [source] Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2006Suvi Ojanperä An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFitÔ) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFitÔ for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17,ppm, respectively, corresponding to 0.65 and 0.60,mTh. For routine screening practice, a SigmaFitÔ tolerance of 0.03 and a mass tolerance of 10,ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFitÔ or mass values. The results show that isotopic pattern matching by SigmaFitÔ is a powerful means of identification in addition to accurate mass measurement. Copyright © 2006 John Wiley & Sons, Ltd. [source] Fifty thousand galaxies at a glanceASTRONOMY & GEOPHYSICS, Issue 3 2002Klaus Meisenheimer Klaus Meisenheimer and Christian Wolf look forward to COMBO-17, a deep sky survey that should make it possible to bridge the gap between matter in the early universe and the pattern of galaxies that we see today. Abstract The COMBO-17 survey should significantly improve our understanding of galaxy evolution during the last 10 billion years in detail, given its multicolour method to determine redshifts and spectral types for 40 000 galaxies within more than a square degree of sky. In addition, mass determination in the supercluster A901/902 should give significant information about the total mass and distribution of dark matter in the universe. In particular, it should be possible to derive new results about the relative distribution of luminous and dark matter. We see this as a significant step in order to answer a decisive question of cosmology: how did the galaxies form from the density peaks in the early universe? [source] Quality control of protein standards for molecular mass determinations by small-angle X-ray scatteringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2 2010Shuji Akiyama Small-angle X-ray scattering (SAXS) is a powerful technique with which to evaluate the size and shape of biological macromolecules in solution. Forward scattering intensity normalized relative to the particle concentration, I(0)/c, is useful as a good measure of molecular mass. A general method for deducing the molecular mass from SAXS data is to determine the ratio of I(0)/c of a target protein to that of a standard protein with known molecular mass. The accuracy of this interprotein calibration is affected considerably by the monodispersity of the prepared standard, as well as by the precision in estimating its concentration. In the present study, chromatographic fractionation followed by hydrodynamic characterization is proposed as an effective procedure by which to prepare a series of monodispersed protein standards. The estimation of molecular mass within an average deviation of 8% is demonstrated using monodispersed bovine serum albumin as a standard. The present results demonstrate the importance of protein standard quality control in order to take full advantage of interprotein calibration. [source] The stellar mass ratio of GK PerseiMONTHLY NOTICES OF THE ROYAL ASTRONOMICAL SOCIETY, Issue 3 2002L. Morales-Rueda We study the absorption lines present in the spectra of the long-period cataclysmic variable GK Per during its quiescent state, which are associated with the secondary star. By comparing quiescent data with outburst spectra we infer that the donor star appears identical during the two states and the inner face of the secondary star is not noticeably irradiated by flux from the accreting regions. We obtain new values for the radial velocity semi-amplitude of the secondary star, , a projected rotational velocity, and consequently a measurement of the stellar mass ratio of GK Per, . The inferred white dwarf radial velocities are greater than those measured traditionally using the wings of Doppler-broadened emission lines suspected to originate in an accretion disc, highlighting the unsuitability of emission lines for mass determinations in cataclysmic variables. We determine mass limits for both components in the binary, and . [source] Modern analytical ultracentrifugation in protein science: A tutorial reviewPROTEIN SCIENCE, Issue 9 2002Jacob Lebowitz Abstract Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein,protein, protein,nucleic acid, and protein,small molecule binding. [source] The empirical upper limit for mass loss of cool main sequence starsASTRONOMISCHE NACHRICHTEN, Issue 4 2008A. Lednicka Abstract The knowledge of mass loss rates due to thermal winds in cool dwarfs is of crucial importance for modeling the evolution of physical parameters of main sequence single and binary stars. Very few, sometimes contradictory, measurements of such mass loss rates exist up to now. We present a new, independent method of measuring an amount of mass lost by a star during its past life. It is based on the comparison of the present mass distribution of solar type stars in an open cluster with the calculated distribution under an assumption that stars with masses lower than Mlim have lost an amount of mass equal to ,M. The actual value of ,M or its upper limit is found from the best fit. Analysis of four clusters: Pleiades, NGC 6996, Hyades and Praesepe gave upper limits for ,M in three of them and the inconclusive result for Pleiades. The most restrictive limit was obtained for Praesepe indicating that the average mass loss rate of cool dwarfs in this cluster was lower than 6 × 10,11 M,/yr. With more accurate mass determinations of the solar type members of selected open clusters, including those of spectral type K, the method will provide more stringent limits for mass loss of cool dwarfs. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] | |||||||||||||||||||||||||