Charles G. Edmonds

Electrospray ionization mass spectrometry.

Publisher Summary This chapter discusses electrospray ionization mass spectrometry. Electrospray ionization (ESI) occurs during the electrostatic nebulization of a solution of charged analyte ions by a large electrostatic field gradient (approximately 3 kV/cm). Highly charged droplets are formed in a dry bath gas, at near atmospheric pressure. These charged droplets shrink as neutral solvent evaporates until the charge repulsion overcomes the cohesive droplet forces leading to a “Coulombic explosion.” Large oligopeptides and small proteins so far successfully examined by ESI-MS show a distribution of multiply charged molecular ions arising (in positive ion mode) by either proton or alkali ion attachment and no evidence of fragment ions because of fragmentation unless dissociation is induced during transport into mass spectrometer vacuum by collisions at higher energy. The largest protein so far examined is the native covalent dimer of bovine serum albumin with a M r of more than 130 kDa. It should be noted that in the case of noncovalently bonded species, such as protein subunits, the mass spectra show only the contributions of the individual subunits. These mass spectra show a distribution of charge states which usually spans about one-half the number of the most abundant charge state.

Collisional activation and collision-activated dissociation of large multiply charged polypeptides and proteins produced by electrospray ionization

Collisional activation (CA) and collision-activated dissociation (CAD) of multiply protonated molecular ions produced by electrospray ionization using an atmospheric pressure source are described. A TAGA 6000E triple-quadrupole mass spectrometer, in both unmodified and differentially pumped inlet arrangements, was used to investigate CA and CAD during transfer through the atmosphere-vacuum interface and subsequent CAD in the tandem instrument. Melittin, which has a molecular weight (Mr) of 2846, is efficiently dissociated in the interface at higher nozzle-skimmer voltages, yielding fragmentation that can be assigned to the various charge states. Selection of such product ions formed in the interface for subsequent tandem mass spectrometry allows confirmation of earlier sequence assignments and extends the utility of these methods. Various charge states of larger polypeptides, such as human parathyroid hormone (1–44) (Mr 5064), can be efficiently collisionally dissociated in the second (rf-only) quadrupole. However, for molecular ions of this size, the low-energy collisions used for CAD yield only partial sequence information. For large molecules such as horse heart myoglobin (Mr 16,951), the effects of nozzle-skimmer bias are explored, and it is shown that higher charge states (at ≤ m/z 1400) can be effectively dissociated in the interface. Initial results for both metastable (unimolecular) and CAD for myoglobin are reported. The potential and limitations of CAD for large biomolecular ions are discussed. The feasibility of fingerprinting for proteins is illustrated by the CAD spectra of cytochrome c from nine species.

Capillary electrophoresis-electrospray ionization-mass spectrometry

Aspects of the innovations presented herein relate to improved systems that in some embodiments perform capillary electrophoresis (CE) and CE in conjunction with electrospray ionization (ESI) as an input to a mass spectrometry system (MS). Some embodiments use a high voltage isolated CE power supply that is configured to float on the high voltage output of an ESI-MS power supply, with a protective resistance in the ESI-MS path, as well as DC/DC converter isolation and communication system isolation for the isolated CE power supply. Some embodiments additionally use a cartridge assembly integrating separation and conductive fluid capillaries with fluid cooling and protective retractable housings for the capillary end portions and for the ESI output. The protective housing may further be used with an adapter for interfacing with different MS systems.

Instrumentation for high-performance capillary electrophoresis—mass spectrometry

Abstract An automated, commercially available capillary electrophoresis (CE) instrument was modified for interfacing with electrospray ionization mass spectrometry (ESI-MS). A Beckman P/ACE 2000 CE instrument with both electrokinetic and hydrostatic sample injection capabilities and efficient temperature control of the separation conditions was selected on the basis of these characteristics for this project. Modifications to it included extension of the fluid circulation path in an interface block extension and electrical modifications due to changes in electrophoresis current measurement capabilities. The capabilities for complete computer automation, capillary temperature control and on-line UV detection were retained. The minimum capillary length was increased by 35 cm due to the extension to the electrospray ionization—mass spectrometer interface. Initial results employing a laboratory—designed liquid sheath electrospray source are presented which show that very high separation efficiencies can be routinely obtained using the modified instrumentation. Capabilities for combined CE-MS are demonstrated for peptide and protein mixtures.

Sensitivity considerations for large molecule detection by capillary electrophoresis-electrospray ionization mass spectrometry

The use of the electrospray ionization (ESI) method for interfacing capillary electrophoresis with mass spectrometry (CE-MS) is particularly well suited for the analysis of large molecules due to the multiple charging phenomenon. While ionization efficiency is very high, the available ion current is dispersed over more peaks so that the maximum peak intensity obtainable declines significantly for large molecules. Sensitivity with ESI can be improved by operation at very low flow-rates, an ideal situation for CE-MS. These and other considerations related to sensitivity are illustrated using ESI-MS measurements for cytochrome c.

Collisional activation and dissociation of large multiply charged proteins produced by electrospray ionization

Abstract Electrospray ionization is capable of producing multiply charged ions of large biopolymers having molecular weights in excess of 100 kDa. Primary structural information can be obtained from highly charged molecular ions by collisionally activated dissociation (CAD) in the atmospheric pressure/vacuum interface of the quadrupole mass spectrometer. Ions are sampled from atmospheric pressure through a 1-mm nozzle orifice to a 2-mm opening skimmer in front of a radiofrequency-only quadrupole. The pressure in the nozzle-skimmer region is typically ≈ 2 Torr. With the potential between the nozzle and skimmer elements (ΔN-S) approximately +185 V, ions due only to intact molecular species are typically observed. Increasing ΔN-S allows for crude control of the collision energy in this region since collision energy depends on the charge state. At ΔN-S > + 300 V, most of the multiply charged molecular ions are dissociated, producing singly and multiply charged product ions. For example, at a ΔN-S value of + 335 V, molecular ions from bovine, human, and rat serum albumin (≈ 580 amino acid residues, Mr ≈ 66 kDa) are efficiently dissociated to yield distinct product ion mass spectra. Series of 3+, 4+, and 5+ “b-type” fragment ions originating from dissociation at amino acid residues ≈ 15–30 from the NH2-terminus of the polypeptide sequence are prominent in the spectra. These results are consistent with other CAD data of smaller proteins showing that fragmentation generally occurs from the ends of the molecule. Extended mass spectrometry (MS)/MS analysis (CAD-MS-CAD-MS or effectively MS-MS-MS) with a triple quadrupole MS apparatus is potentially available for confirmation of product ion assigments and for obtaining sequence information from molecular regions unprobed by the initial CAD step for the intact molecular ion.

Electrospray ionization mass spectrometric characterization of acrylamide adducts to hemoglobin

The most common procedure to identify hemoglobin adducts has been to cleave the adducts from the protein and characterize the adducting species, by, for example, derivatization and gas chromatography/mass spectrometry. To extend these approaches we used electrospray ionization mass spectrometry (ESI-MS) to characterize adducted hemoglobin. For this we incubated [14C]acrylamide with the purified human hemoglobin (type A0) under conditions that yielded high adduct levels. When the hemoglobin was separated by reversed-phase high-performance liquid chromatography (HPLC), 65% of the radioactivity copurified with the beta-subunit. Three adducted species were prominent in the ESI mass spectrum of the intact beta-subunit, indicating acrylamide adduction (i.e., mass increase of 71 Da) and two additional unidentified moieties with mass increments of 102 and 135 Da. Endoproteinase Glu-C digestion of the adducted beta-subunit resulted in a peptide mixture that, upon reversed-phase HPLC separation, provided several radiolabeled peptides. Using ESI-MS we identified these as the V91-101 and V102-122 peptides that represent the cysteine-containing peptides of the beta-subunit. These results provide definitive information on acrylamide-modified human hemoglobin and demonstrate that ESI-MS provides valuable structural information on chemically adducted proteins.

Sesquiterpenes in leaf pocket resins of Copaifera species

Abstract Four sesquiterpene leaf resin components were isolated and identified from Copaifera leaf resin. Additional GC and mass spectrometric evidence support the close similarity of Copaifera leaf pocket resin composition with that of the related genus, Hymenaea .

Characterization of radiation-induced thymine-tyrosine crosslinks by electrospray ionization mass spectrometry

Exposure to ionizing radiation leads to formation of covalent crosslinks between DNA and proteins. The nature, extent and site of the modifications are not well understood due to the difficulty in assessing free radical-induced damage in biopolymers. Electrospray ionization mass spectrometry (ESI-MS) permits direct analyses of intact oligopeptides, permitting characterization of the radiation-induced DNA-protein covalently crosslinked constituents. Our first application of this methodology to free radical-induced damage was in a model system where angiotensin, a small 10-amino acid peptide, is irradiated at various doses in the presence of excess thymine. The relative yield of crosslinks, which ranged from 0.1 to 15%, was linearly related to radiation dose for doses from 0.1 to 100 Gy. Detection of thymine-tyrosine moieties in this model system was possible at doses as low as 0.1 Gy with a signal-to-noise ratio of 4 to 1. ESI-MS revealed that the site of crosslink was located exclusively on the tyrosine residue as expected.

Combined Liquid Chromatography-Mas Spectrometry for Microscale Structural Studies of Carbohydrates

Abstract Combined HPLC-thermospray was spectrometry of mono-and disaccharides, 1-0-methylglycosides and 0-permethy mono - through tetrasaccharicdes has been studied to asess the potential role of thermospray ionization for microscale structural studies of saccharides and glycoconjugates, and for high sensitivity detection of liquid chromatographic effluents. Using NH4CO2H as evaluant for reversed-phase HPLC, abundant MNH4 4 ions are formed form monosaccharides and mono-and per-methylated saccharides, and are suitable formonitoring sub-nanogram constituents in HPLC effluents. Detection of 100 pg (0.5pmole) of 1-0-methylhexopy-ranosides with signal/noise> 10 is demonstrated.