Showing papers in "Rapid Communications in Mass Spectrometry in 1991"

Conformational changes in proteins probed by hydrogen‐exchange electrospray‐ionization mass spectrometry

Abstract: Hydrogen-exchange electrospray-ionization mass spectrometry is demonstrated to be an effective new method for probing conformational changes of proteins in solutions. The method is based on the mass spectrometric measurement of the extent of hydrogen/deuterium exchange that occurs in different protein conformers over defined periods of time. Results are presented in which hydrogen-exchange electrospray-ionization mass spectrometry is used to probe conformational changes in bovine ubiquitin induced by the addition of methanol to aqueous acidic solutions of the protein.

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Abstract: Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.

Metastable decay of peptides and proteins in matrix‐assisted laser‐desorption mass spectrometry

Abstract: Fragmentation of protein and peptide ions generated by matrix-assisted laser desorption has been investigated using a modified LAMMA 1000 reflecting time-of-flight (TOF) mass spectrometer. Whereas fragmentation of covalent bonds prior to ion acceleration (i.e., within several ns after the laser pulse) in general is not observed using the matrix technique, extensive fragmentation on a longer time scale can be studied in our instrument. The high mass resolution (M/ΔM≈1200–1800 for insulin and peptides) permits the investigation of even small mass losses from parent molecular ions (occuring in the first section of the field-free drift region) by measuring flight time differences of daughter ions acquired during passage through a two-stage reflection. The kind and extent of this metastable decay has been found to depend strongly on the substance under investigation. Typical fragmentations are loss of ammonia and parts of the amino acid side-chains. The large abundances of peaks due to such metastable fragmentation, observed for most of the peptides and proteins investigated, may, at least in part, explain peak broadening (and, hence, poor mass resolution) typical in matrix-assisted laser-desorption TOF mass spectra.

Improved electrospray ionization of synthetic oligodeoxynucleotides

Abstract: A method is described for rapid purification of synthetic oligodeoxynucleotides to remove sodium counter ions prior to electrospray ionization mass spectrometry. The oligomers, following purification by gel electrophoresis, are precipitated from ammonium acetate to replace sodium ions by ammonium ions, and dissolved in water. Negative-ion electrospray spectra are presented for oligomers with up to 48 residues in which the most intense peaks correspond to the [MnH]n− ion. The spectrum of 77-mer (Mr24039) shows the predominant peak as due to retention of only a single sodium ion. Changes in the spectra are reported for different instrument orifice voltages and different solution pH. The method should permit rapid, accurate analysis of most typical, synthetic oligodeoxynucleotides.

Maximum entropy deconvolution in electrospray mass spectrometry

Abstract: The maximum entropy (MaxEnt) technique has been well established in image processing for the last decade. In more recent times MaxEnt has been applied to spectroscopic techniques and shows considerable promise. As part of a much wider evaluation of the Cambridge software, specifically MemSys3, the MaxEnt technique has been successfully applied to electrospray mass spectra.

Properties of matrix-assisted laser desorption. Measurements with a time-to-digital converter.

Abstract: Some properties of matrix-assisted laser desorption have been studied using single-ion-counting methods and a time-to-digital converter. The methods allow examination of the process for irradiances near the reported threshold for observation with a transient recorder. All measurements were made using bovine insulin as a test compound. We present direct evidence that an irradiance threshold near 10(6) W cm-2 exists for ion production, and that the process is a collective effect, either involving a large number of molecular ions (approximately 10(4) in a successful event or none at all. Above the threshold, the yield is found to scale with a high power (4th to 6th) of the irradiance. Measurements of initial velocity distributions indicate an axial velocity spread corresponding to approximately 50 eV and a radial velocity spread corresponding to approximately 2.4 eV. Thus the ejection or extraction mechanism appears to be strongly asymmetric.

Massive cluster impact mass spectrometry: A new desorption method for the analysis of large biomolecules

Abstract: A new ion desorption method is described that utilizes a primary beam of massive, multiply charged cluster ions to generate secondary ions of peptides in a glycerol matrix. The massive cluster ion beam is generated via electrohydrodynamic emission using a 1.5 M solution of ammonium acetate in 30% aqueous glycerol. Negative ion spectra of peptides obtained using this technique show greatly decreased relative intensities for fragment ions and 'chemical noise' background when compared to spectra obtained using a xenon atom primary beam. The near absence of fragments derived from radiation damage to the sample solution is attributed to the impact of primary particles with energies less than 1 eV/nucleon.

Molecular weight determination of a polyamidoamine Starburst polymer by electrospray ionization mass spectrometry

Abstract: Electrospray ionization mass spectrometry has been demonstrated to be a useful technique for the molecular weight determination of a Starburst polymer. Mass spectra of multiply charged ions are deconvoluted to provide molecular weight information of many components in a non-separated mixture. These data may also be used for determining a polydispersity value for this systhetic polymer system.

Investigation of collisional‐activation decomposition process and spectra in the transport regions of an electrospray single‐quadrupole mass spectrometer

Abstract: The use of collisional-activation dissociation (CAD) in the electrospray transport region was evaluated for generating structural information on several pesticides and antibiotics. The collision energy used to generate the CAD spectra could be varied easily by changing the capillary/skimmer potential difference, imparting from 0 eV to above 16 eV internal energy to the near thermal ions generated by electrospray. The internal energy distribution for low-energy collisions (capillary/skimmer potential difference of 20 V) closely matches the curves generated by a triple-quadrupole mass spectrometer. Furthermore, the CAD spectra for selected compounds generated by electrospray in the transport region at a capillary/skimmer potential difference of 30-50 V closely resembled those obtained from the [M + H]+ ion by a triple quadrupole using 30 eV collision energy. The CAD of ions in the transport region resulted in 70% to 80% daughter-ion yields and minimal loss in overall ion current compared to the ion current for protonated or cationized parent molecules. The major daughter ions for 10 pg of Aldicarb and penicillin G could be detected (signal-to-noise ratio greater than 5) under full-scan CAD conditions.

High temperature mass spectrometry in materials research

Abstract: Vaporization is an important property in materials research since it limits the use of the materials at high temperatures and since valuable thermodynamic data can be determined from vapour pressures. High-temperature mass spectrometry is the most versatile method for the elucidation of vaporization processes. Numerous results have been obtained. This article describes the fundamentals of the method and gives examples of its application. These examples describe the determination of thermodynamic properties for the gaseous species and condensed phases

Thermally induced dissociation of ions from electrospray mass spectrometry

Abstract: The thermally induced dissociation (TID) of inos at near atmospheric pressure, followed by their mass spectraometric analysis, is demonstrated The TID occurs in a heated metal capillary interface, and should be broadly applicable to any system capable of producing ions at high pressure The heated capillary provides a reaction vessel, potentially very useful for kinetic and mechanistic studies of ions The technique has been applied to the fragmentation of large multiply charged ions from an electrospray ionization source Preliminary results show that stability for multiply protonated petide or protein molecules in inversely related to its charge

Electrospray ionization for analysis of platelet-activating factor.

Abstract: Platelet-activating factor (PAF) was analyzed by electrospray-ionization spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na−59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M + Cl]−. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.

Analysis of peptides and proteins by capillary electrophoresis/mass spectrometry using acidic buffers and coated capillaries.

Abstract: The direct coupling of capillary electrophoresis (CE) and mass spectrometry, combined with ionspray ionization using a coaxial capillary arrangement, is described. The CE/mass spectrometer interface is shown to be effective for the analysis of native and tryptic peptides and of proteins of high molecular weight such as bovine serum apotransferrin (approximately 78 kDa). Adsorption of cationic analytes under acidic buffer conditions is minimized through the use of a non-covalent coated capillary possessing an overall positive charge. Since the direction of the electroosmotic flow is thus reversed, compared to that in conventional CE separation on uncoated capillaries, migration of cations is achieved by applying a negative voltage (typically -30 kV) at the injector end of the capillary. In addition to the inherent advantage of providing pre-formed cationic species for mass spectral detection, this arrangement permits analysis of proteins of high isoelectric points even at low pH. The ability to conduct electrophoresis of globular proteins under acidic conditions also provides a means of monitoring their conformational changes, as reflected both by the variation of migration times and by concurrent changes in the multiply charged ion envelopes.

The ion-trap mass spectrometer in ion structure studies. The case of [MH]+ ions from chalcone

Abstract: Ion-structure investigation of the [MH]+ ions from chalcone has been carried out by collision experiments performed with an ion-trap mass spectrometer (ITMS), operating with the application of a supplementary RF voltage. The data so obtained, together with those achieved by comparison with labelled model compounds, have led to a clear identification of the [MH]+ ion structure, proving the high potentialities of the ITMS in such a field.

High mass-resolution using a quadrupole ion-trap mass spectrometer

Abstract: Modifications have been made to the quadrupole ion-trap mass spectrometer (ITMS) to improve the resolution. An offset-digital-to-analog converter/attenuator network is used to slow down the scan rate in the mass-selective instability mode of operation with reasonance ejection. Attenuations of the scan rate by 200 to 700 times increase the resolution from 500 (with no attenuation) to 40 000 and 100 000 (full width at half maximum), respectively, for the [M + H]+ peak of substance p (m/z 1348). These improved resolutions are achieved in experiments which use resonance ejection to extend the mass-to-charge range.

Charge stripping C60

Abstract: Measurements of the translational energy loss accompanying the charge-stripping reactions M + +N→M 2+ +N+e − and M 2+ N→M 3+ +N+e − have been performed for C 60 + , C 70 + and C 60 2+ , C 70 2+ , respectively. The energy necessary to remove the second electron from Buckminsterfullerene was determined

A collisional study of some C60 and C70 fullerene ions

Abstract: The high-energy collision-induced dissociation (CID) mass spectra of C, C, C, C and C are reported. In all cases, fragmentation occurs by loss of an even number of carbon atoms, in agreement with photodissociation studies of C and C. No charge-separation reactions are observed for the multiply charged ions. Collision-induced dissociation of C leads to a slight preference for formation of C, while dissociation of C and C shows a more pronounced preferenced for formation of C and C, respecstively. Under electron-capture Chemical-ionization conditions the fullerenes readily capture an electron to form the molecular anions. Collision-induced dissociation of C and C leads to elimination of 1, 2 and 4 carbon atoms; the elimination of one carbon is unique among larger carbon cluster ions. Charges inversion of the molecular anions leads to formation of the respective molecular cations and fragementation of these cations by loss of an even number of carbon atoms. Formation of C is slightly preferred in the C case, while C is the most abundant ion in the charge inversion mass spectrum of C.

Simple physical models for coulomb-induced frequency shifts and coulomb-induced inhomogenous broadening for like and unlike ions in fourier transform ion cyclotron resonance mass spectrometry

Abstract: Two simple models which Correspond more closely to physical reality than some prior models are proposed to account for the Coulomb-induced frequency shifts to lower frequency, Which have been observed in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. The first model consists of two different-mass point charges which undergo cyclotron orbits with the same orbit centers at their respective cyclotron frequencies. The model predicts that each excited cyclotron motion should induce a negative frequency shift in the other's cyclotron motion. The model predicts a zero frequency shift for ions of the same mass. The physical basis for the Coulomb-induced shift is most easily seen in a coordinate frame which rotates at the cyclotron motion of the ion whose frequency is shifted. It is noted that a prior model for Coulomb-induced frequency shifts is more appropriate for scanning ICR than FT-ICR and the reason is described for the correctness of the predictions of this less appropriate model. A second model, the line model, is created by extension of the point model. The line model gives rise to a position-dependent frequency shift which is synonymous with inhomogeneous Coulomb broadening. When a non-quadrupolar electrostatic trapping field is included in the model, ‘like’ ions become ‘unlike’ and Coulomb-shifting and Coulomb-broadening apply, as for ‘like’ ions. The analysis predicts that ICR cells which are designed to have negligible trapping fields over most of their volume will be less sensitive to Coulomb shifting/broadening than ordinary ICR cells.

Matrix/sample interactions in ultraviolet laser-desorption of proteins

Abstract: Examination, by optical microscopy, of the matrix/analyte morphology of a sample propared from a droplet in matrix-assisted UV laser-desorption mass spectrometry (MUVLDMS) reveals that the matrix crystallizes to a large extent, thus altering the concentration of the material in regions on the surface from that of the bulk applied solution. Microscopy reveals that vanillic acid and to a lesser extent nicotinic acid matrices form prominent crystalline rings around the dried sample spot while sinapic acid forms a more uniform dispersion of the crystallized matrix. Insulin analyte investigated in this report tendss to form a thin isotropic phase in the center of the drops. The first reported use of a KrF excimer laser (249 nm) in MUVLDMS is also discussed.

Facile assignment of sequence ions of a peptide labelled with 18O at the carboxyl terminus

Abstract: The combination of collision-induced dissociation (CID) and linked-scan analysis was used for analysing the sequence ions from the precursor ion of a peptide, which had been labelled with 18O at its carboxyl terminus (C-terminus) using 40 atom % H218O. The CID and linked-scan mass spectrum of the labelled peptide gave two series of sequence-ion signals: the one, originating from the C-terminus of the labelled peptide, showed a doublet signal due to the part-incorporation of 18O into the carboxyl group at the C-terminus, while the other, originating from the amino terminus (N-terminus), has the natural isotopic ion distribution. From the distribution of the isotopic ions in a single CID spectrum, the sequence ions containing the C-terminus could be readily differentiated from those containing the N-terminus, allowing the facile assignment of sequence ions to the amino-acid sequence of peptide by CID and linked-scan analysis. This method was successfully applied to determination of the amino-acid sequence of the light-chain of mouse anti-porphyrin monoclonal antibody.

Laser-pulse ejection of organic molecules from a matrix : lessons from fast-ion-induced ejection

Abstract: Lessons learned from electronic sputtering by incident ions of condensed-gas solids and solids composed of biomolecules are applied to laser-pulse-induced ejection of biomolecules inbedded in a matrix of small molecules. In electronic sputtering by fast ions, single excitation desorption, thermally activated sputtering, and pressure-pulse sputtering have been observed. In addition, yields for ejected ions have been shown to exhibit trends different from yields for ejected neutrals. The parallel processes are considered for laser-pulse ejection of a martix with imbedded molecules and expressions are given to examine the yield data.

Fast protein sequence determination with matrix-assisted laser desorption and ionization mass spectrometry.

Abstract: Measurement and identification of digested peptides by matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS) is demonstrated. Synthetic human parathyroid hormone, pTH (1-34), with a molecular mass of 4117.8 Da was digested with carboxypeptidases Y and B and the sequence of 14 amino acids from the C-terminus of the peptide was determined by analyzing the molecular mass of the truncated peptides. Furthermore, a tryptic digestion of pTH (1-34) was carried out and a molecular mass map of pTH (1-34) was obtained. With the results of the proteolytie digestion a rapid confirmation of the amino-acid sequence of the protein was possible. It is shown that the results of the tryptic digestion can be used for the unambiguous identification of the amino acid residues Lys and Arg, which cannot be distinguished with a mass spectrometer because of their equal nominal masses. Several advantages of amino acid sequence determination by the combination of digestion and LDI-MS are obvious: high sensitivity in the low pmol range, fast digestion time due to high enzyme/substrate ratios, quantification is unnecessary because the amino acids are identified by their molecular mass differences, the low chemical expenditure for the digestions and the accuracy of the sequence determination. Measurements with LDI-MS are fast: sample preparation and the measurement take only a few min. The mass determination and amino acid sequence is completely unimpaired by amino acid contaminations or impurities in the sample. The sensitivity of the method is in the low pmol to fmol range and thus comparable to other analytical methods. The amino-terminal sequence determination of peptides by the Edman procedure is nowadays a standard and routine method. However with the development of new mass spectrometric and ionization methods such as fast-atom bombardment mass spectrometry (FAB-MS) plasma desorption mass spectrometry (PD-MS),4.s electrospray-mass spectrometryGx and recently, matrix-assisted laser desorption and ionization mass spectrometry (LDI-MS),”I3 novel analytical tools have become available. They offer interesting opportunities for biochemists to determine amino acid sequences of peptides and proteins. The amino acid sequence of a peptide can be revealed by measuring the molecular mass of degraded peptides of an enzymatic digestion instead of determining the released amino acids. FAB-MS and PDMS have already been used to establish the sequence or to elucidate the primary structure of peptides with carboxypeptidase digestion. 142’ To determine the amino acid structure of peptides and proteins, the mass spectrometric method must fulfill several requirements. It must be possible to generate singly-charged molecular ions from a mixture of different proteins. No fragmentation should occur and only low amounts of higher-charged ions should be detectable, otherwise this can lead to very complex mass spectra where the assignment of the ion signals is difficult or impossible. The accuracy of the mass determination has to be better than 1 Da. The primary amino acid residues Ile and Leu have identical masses and Lys and Gln have the same nominal mass. These amino acids cannot be separated by mass spectrometric means alone. Leu/Asn and Lys/Glu differ only by 0.94 Da in their molecular masses. If the mass can be * Author to whom correspondence should be addressed determined with the above mentioned precision these two amino acid pairs and all other primary amino acids should be distinguishable. Furthermore, the resolution of the mass spectrometer has to be high enough to resolve subsequent peptide fragments. Gly has the lowest nominal residual mass (57Da) of the primary amino acids. This number defines the mass difference between two ion signals which must be resolvable by the mass spectrometric method. In this paper we present the analysis of enzymatic digestions of a peptide by matrix-assisted laser desorption and ionization mass spectroscopy (LDI-MS). Although this method was developed only 3 years ago by Hillenkamp and Karas’ it has already shown its potential as an easy-to-use mass spectrometric method for the highly accurate determination of molecular masses of large biomolecules.”’3 Due to the high sensitivity, the dynamic range of better than 1: 100, the almost universal selectivity and the complete absence of fragmentation, the method can be used especially for the mass determination of peptides contained in complex mixtures. LDI-MS completely fulfils the above mentioned requirements for a mass spectrometric method. The best resolution which can be achieved with our system is mlhm = 400-500 (50% valley). Thus peptides up to a mass of 22800Da can be resolved if they differ only by one Gly amino acid. The accuracy of the mass determination obtained is typically +0.03%. For peptides with masses higher than 3000 Da this error might lead to ambiguous results in identification of the cleaved amino acid residues. However if several fragments can be detected in the same measurement, the error in mass determination can be lowered because systematic errors are cancelled out by calculating mass differences. Since the work by Hayashi2’ and K~hn,?~ carboxy

Factors affecting the ionization efficiency of quaternary ammonium compounds in electrospray/ionspray mass spectrometry

Abstract: A series of quaternary ammonium salts was analysed by the ionspray technique under different conditions in order to study the influence of some important factors on the ionization efficiency. The factors taken into account were droplet charging, sample ion, counter ion, solvent composition, and concentration. Measurement of the spray current leaving the nebulizer has shown that the spray current increases linearly with sample concentration up to at least 50 pmol/mu-L. The measured spray current is approximately 30% of the calculated current corresponding to a given concentration of quarternary ammonium ions in solution leaving the nebulizer at a given flow rate. The results obtained show that tetraalkylammonium salts give the best sensitivity with no influence of sample-ion shape or counter ion. The introduction of other functional groups in the quaternary ammonium ion decreases the sensitivity. The dynamic range for full-scan mass spectra of the compounds studied appears to be linear for concentrations from 0.1 pmol/mu-L (10(-7) M) up to 10 pmol/mu-L (10(-5)M).

Analysis of polycyclic aromatic compounds by supercritical fluid charomatography/mass spectrometry using atmospheric-pressure chemical ionization

Abstract: The development of microbore packed-column supercritical fluid chromatography/atmospheric pressure chemical ionization mass spectrometry (SFC/APCI-MS) for the analysis of polycyclic aromatic compounds (PAC) is described. The SCF system was coupled to the mass spectrometer using an improved interface designed to accommodate commercially available fused silica restrictors. The atmospheric pressure chemical ionization characteristics of PAC are discussed and the use of chemical ionization reagents to modify the type of spectra obtained is described

An ion-storage time-of-flight mass spectrometer for analysis of electrospray ions

Abstract: A preliminary design and implementation of a novel approach to electrospray-mass spectrometry anr described. Based on a time-of flight mass analysis, the instrument provides several important advantages for on-line mass analysis: 1, simplicity, ease of use and low manufacturing cost; 2, rapid scan speed, yielding quasi-instantaneous full mass scans at repetition rates up to several kHz; 3, soft ionization and accurate mass determination of extremely large analyte molecules; 4, high sensitivity.

Matrix-assisted laser desorption ionization with a magnetic mass spectrometer.

Abstract: Matrix-assisted laser desorption ionization has been carried out with a high mass double-focusing magnetic mass spectrometer. The pulsed ion signal, generated by irradiation of the sample (substance P, ubiquitin, and cytochrome c) embedded in 2,5-dihydroxybenzoic acid with a XeF excimer laser (353 nm, 12 ns pulse, 10-160 Hz), was recorded with an integrating array detector. Good resolution of 2600 (full width at half maximum) and high sensitivity (a few pmol) were obtained. The loss of small neutral fragments was observed, supporting the notion that the peak broadening observed in the time-of-flight mass spectrometers commonly used for this ionization mode is due to such metastable decomposition.