High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer

A novel electrospray ionization/Fourier transform ion cyclotron resonance mass spectrometer based on a 7-T superconducting magnet was developed for high-resolution accurate mass measurements of large biomolecules. Ions formed at atmospheric pressure using electrospray ionization (ESI) were transmitted (through six differential pumping stages) to the trapped ion cell maintained below 10^?9 torr. The increased pumping speed attainable with cryopumping (> 10⁵ L/s) allowed brief pressure excursions to above 10^?4 torr, with greatly enhanced trapping efficiencies and subsequent short pumpdown times, facilitating high-resolution mass measurements. A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ES1 source and were open only during ion injection. Coupled with the use of the pulsed-valve gas inlet, the trapped ion cell was generally filled to the space charge limit within 100 ms. The use of 10–25 ms ion injection times allowed mass spectra to be obtained from 4 fmol of bovine insulin (M_r 5734) and ubiquitin (M_r 8565, with resolution sufficient to easily resolve the isotopic envelopes and determine the charge states. The microheterogeneity of the glycoprotein ribonuclease B was examined, giving a measured mass of 14,898.74 Da for the most abundant peak in the isotopic envelope of the normally glycosylated protein (i.e., with five mannose and two N-acetylglucosamine residues (an error of approximately 2 ppm) and an average error of approximately 1 ppm for the higher glycosylated and various H₃PO₄ adducted forms of the protein. Time-domain signals lasting in excess of 80 s were obtained for smaller proteins, producing, for example, a mass resolution of more than 700,000 for the 4⁺ charge state (m/z 1434) of insulin.     

Gas‐phase formation of protonated benzene during collision‐induced dissociation of certain protonated mono‐substituted aromatic molecules produced in electrospray ionization

Protonated benzene, C₆H, has been studied extensively to understand the structure and energy of a protonated organic molecule in the gas phase. The formation of C₆H is either through direct protonation of benzene, i.e., chemical ionization, or through fragmentation of certain radical cations produced from electron ionization or photon ionization. We report a novel observation of C₆H as a product ion formed in the collision‐induced dissociation (CID) of protonated benzamide and related molecules produced via electrospray ionization (ESI). The formation of C₆H from these even‐electron precursor ions during the CID process, which has not been previously reported, is proposed to occur from the protonated molecules via a proton migration in a five‐membered ring intermediate followed by the cleavage of the mono‐substituent C? C bond and concurrent formation of an ion‐molecule complex. This unique mechanism has been scrutinized by examining some deuterated molecules and a series of structurally related model compounds. This finding provides a convenient mean to generate C₆H, a reactive intermediate of considerable interest, for further physical or chemical investigation. Further studies indicate that the occurrence of C₆H in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) appears to be a rather common phenomenon for many compounds that contain ‘benzoyl‐type’ moieties. Hence, the observation of the C₆H ion in LC/ESI‐MS/MS can be used as an informative fragmentation pathway which should facilitate the identification of a great number of compounds containing the ‘benzoyl‐type’ and similar structural features. These compounds are frequently present in food and pharmaceutical products as leachable impurities that require strict control and rapid elucidation of their identities. Copyright © 2010 John Wiley & Sons, Ltd.     

Reduction of nitriles to amines in positive ion electrospray ionization mass spectrometry

Some compounds readily form [M+46]+ adduct ions during positive ion electrospray ionization mass spectrometry ((+)ESI-MS) analysis. These [M+46]+ ions were characterized as [M+CH3CH2NH2+H]+ by accurate mass determination. Ethylamine involved in the adduct was proposed to be the reduction product of acetonitrile and this was confirmed using deuterated acetonitrile. Other nitrile-containing compounds tested, including isobutyronitrile and benzonitrile, also formed the adduct ions of the respective amine forms under (+)ESI-MS conditions. Hydrogen/deuterium exchange experiments demonstrated that the reductive hydrogen originated from water. Reduction of nitriles (R-CN) to their respective amines (R-CH2NH2) under (+)ESI-MS conditions expands the ability to identify nitrile-containing chemical unknowns.     

Desorption electrospray ionization tissue imaging using heated nebulizing gas and high‐resolution accurate mass spectra

A new method for tissue imaging using desorption electrospray ionization (DESI) mass spectrometry is described. The technique utilizes a DESI source with a heated nebulizing gas and high‐resolution accurate mass data acquired with an LTQ‐Orbitrap mass spectrometer. The two‐dimensional (2D) automated DESI ion source creates images using the ions that are collected under high‐resolution conditions. The use of high‐resolution mass detection significantly improves the image quality due to exclusion of interfering ions. The use of a heated nebulizing gas increases the signal intensity observed at lower gas pressure. The technique developed is highly compatible with soft tissue imaging due to the minimal surface destruction. Copyright © 2010 John Wiley & Sons, Ltd.     

Chiral analysis of amphetamine-type stimulants using reversed-polarity capillary electrophoresis/positive ion electrospray ionization tandem mass spectrometry

Reversed-polarity (RP) capillary electrophoresis/positive ion electrospray ionization mass spectrometry (CE-ESI+ MS) and tandem mass spectrometry (MS/MS) were utilized for simultaneous chiral separation of nine amphetamine-type stimulants (ATS) (dl-norephedrine, dl-norpseudoephedrine, dl-ephedrine, dl-pseudoephedrine, dl-amphetamine, dl-methamphetamine, dl-methylenedioxyamphetamine, dl-methylenedioxymethamphetamine, and dl-methylenedioxyethylamphetamine). Using highly sulfated gamma-cyclodextrin (SU(XIII)-gamma-CD) as a chiral selector, the nine ATS were completely separated within 50 min. The migrated ATS-CD complex was dissociated at the ESI interface, and only ATS molecules went into the MS detector so that all 18 individual enantiomers were identified by their mass spectra. The detection limit of MS/MS was 10 times more sensitive than those for single MS. Seized d-methamphetamine hydrochloride samples dissolved at high concentration (20 mg/mL) were analyzed. Impurities originating in the precursor such as l-ephedrine and d-pseudoephedrine were detected and identified by tandem mass spectra.     

Enantiomeric differentiation of β‐amino alcohols under electrospray ionization mass spectrometric conditions

The enantiomeric differentiation of a series of chiral β‐amino alcohols (A) is attempted, for the first time, by applying the kinetic method using L‐proline, L‐tryptophan, 4‐iodo‐L‐phenylalanine or 3, 5‐diiodo‐L‐tyrosine as the chiral references (Ref) and Cu²⁺ or Ni²⁺ ion (M) as the central metal ion. The trimeric diastereomeric adduct ions, [M+(Ref)₂+A‐H]⁺, formed under electrospray ionization conditions, are subjected for collision‐induced dissociation (CID) experiments. The products ions, formed by the loss of either a reference or an analyte, detected in the CID spectra are evaluated for the enantiomeric differentiation. All the references showed enantiomeric differentiation and the R_chiral values are better for the aromatic alcohols than for aliphatic alcohols. Notably, the R_chiral values of the aliphatic amino alcohols enhanced when Ni²⁺ is used as the central metal ion. The experimental results are well supported by computational studies carried out on the diastereomeric dimeric complexes. The computational data of amino alcohols is correlated with that of amino acids to understand the structural interaction of amino alcohols with reference molecule and central metal ion and their role on the stabilization of the dimeric complexes. Application of flow injection MS/MS method is also demonstrated for the enantiomeric differentiation of the amino alcohols. Copyright © 2014 John Wiley & Sons, Ltd.     

Sensitive analysis of metal cations in positive ion mode electrospray ionization mass spectrometry using commercial chelating agents and cationic ion‐pairing reagents

Metals play a very important role in many scientific and environmental fields, and thus their detection and analysis is of great necessity. A simple and very sensitive method has been developed herein for the detection of metals in positive ion mode ESI‐MS. Metal ions are positively charged, and as such they can potentially be detected in positive ion mode ESI‐MS; however, their small mass‐to‐charge (m/z) ratio makes them fall in the low‐mass region of the mass spectrum, which has the largest background noise. Therefore, their detection can become extremely difficult. A better and well‐known way to detect metals by ESI‐MS is by chelating them with complexation agents. In this study eleven different metals, Fe(II), Fe(III), Mg(II), Cu(II), Ru(III), Co(II), Ca(II), Ni(II), Mn(II), Sn(II), and Ag(I), were paired with two commercially available chelating agents: ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS). Since negative ion mode ESI‐MS has many disadvantages compared to positive ion mode ESI‐MS, it would be very beneficial if these negatively charged complex ions could be detected in the positive mode. Such a method is described in this paper and it is shown to achieve much lower sensitivities. Each of the negatively charged metal complexes is paired with six cationic ion‐pairing reagents. The new positively charged ternary complexes are then analyzed by ESI‐MS in the positive single ion monitoring (SIM) and single reaction monitoring (SRM) modes. The results clearly revealed that the presence of the cationic reagents significantly improved the sensitivity for these analytes, often by several orders of magnitude. This novel method developed herein for the detection of metals improved the limits of detection (LODs) significantly when compared to negative ion mode ESI‐MS and shows great potential in future trace studies of these and many other species. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.     

The distinction of underivatized monosaccharides using electrospray ionization ion trap mass spectrometry

A convenient method for distinguishing underivatized isomeric monosaccharides has been established using electrospray ionization ion trap mass spectrometry (ESI-ITMS). Mass spectra of hexoses (glucose, galactose, and mannose), N-acetylhexosamines (N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine) and hexosamines (glucosamine, galactosamine, and mannosamine) dissolved in solvent containing 1 mM ammonium acetate were obtained in the positive ion mode. Glucose was distinguished from galactose and mannose in the MS(2) spectrum of the [M+NH(4)](+) ion at m/z 198. The MS(3) spectra generated from [M+NH(4)-H(2)O-NH(3)](+) at m/z 163 showed that galactose and mannose could be distinguished by the ratio of peak intensities at m/z 145 and 127, while the three N-acetylhexosamine and hexosamine stereochemical isomers could be identified by the relative abundance ratios of product ions observed in MS(3) spectra. The investigation of MS and MS(2) spectra from complexes of these monosaccharides with Na(+) and Pb(2+) failed to distinguish these monosaccharide isomers. Therefore, multiple stage mass analysis by ESI-ITMS using either [M+NH(4)](+) or [M+H](+) was useful to distinguish between the isomers of monosaccharides.