High-mass analysis using quadrupolar excitation/ion cooling in a Fourier transform mass spectrometer.

Quadrupolar axialization has been applied to matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of singly charged polymer and protein ions. When a quadrupolar excitation is applied in the presence of argon collision gas, ions are driven to the center of the cell, where they can traverse the conductance limit and be detected in the lower pressure analyzer cell of a dual cell instrument. It is shown that single frequency quadrupolar excitation permits highly selective detection of single oligomers of poly(ethylene glycol) 6000. Resolution enhancement is demonstrated for bovine insulin and ubiquitin, where spectra with resolving powers of 92,000 and 25,000, respectively, are obtained. Finally, quadrupolar axialization is demonstrated to be effective for molecules with masses as large as 13,000 Da.

Structure of the novel heme adduct formed during the reaction of human hemoglobin with BrCCl3 in red cell lysates.

It was previously shown that the reductive debromination of BrCCl3 to trichloromethyl radical by human hemoglobin leads to formation of dissociable altered heme products, two of which are identical to those formed from myoglobin and one which is novel. In this study, we have elucidated the structure of this novel adduct with the use of mass spectrometry, as well as 1H and 13C NMR as a substitution product of a -C(Cl) = CCl2 moiety for a beta-hydrogen atom on the prosthetic heme's ring I vinyl group. From studies with the use of 13C-enriched BrCCl3, it was determined that the added carbon atoms were derived from 2 eq of BrCCl3. A mechanism that involves multiple reductive events and a radical cation heme intermediate is proposed. Consistent with this mechanism, cellular reductants were found to selectively enhance the amount of this novel dissociable heme adduct. These studies reveal fine differences between myoglobin and hemoglobin in the accessibility of reactive intermediates to the ring I vinyl group, as well as the potential importance of cellular reductants on the course of heme alteration.

Ultrahigh resolution matrix-assisted laser desorption/ionization of small proteins by Fourier transform mass spectrometry.

Recent research has demonstrated that matrix-assisted laser desorption/ionization (MALDI) is feasible for Fourier transform mass spectrometric analysis of biomolecules with masses in excess of 50,000 Da. Here, the effects of electrostatic deceleration times and laser energy upon mass resolution are reported. It is demonstrated that optimum deceleration times for singly-charged MALDI-generated protein ions ranging in mass from 2627 to 29,000 Da are a linear function of m1/2 when a 9.5-V decelerating potential is used. Furthermore, higher resolution is obtained with laser fluences close to the threshold for MALDI. Slow metastable decay of molecular ions in the absence of co-matrix is demonstrated for melittin and bovine insulin. It appears that the resolution enhancing effect of co-matrix may result from slowing molecular ion unimolecular decomposition rates sufficiently to allow infrared emission to compete with metastable decay, thus providing the requisite population of long-lived ions for high mass resolution. A spectrum of bovine insulin molecular ion with mass resolution of 30,000 is presented, together with several spectra of lower mass proteins with mass resolution in excess of 100,000. Detection of a doubly-charged carbonic anhydrase trimer ion with a mass of 87,000 Da is reported.

Investigation of a "screened" electrostatic ion trap for analysis of high mass molecules by fourier transform mass spectrometry.

With the recent adaptation of matrix-assisted laser desorption/ionization (MALDI) of large biomolecules and polymers to Fourier transform mass spectrometric analysis, it is of interest to explore the possible advantages of using a "screened" electrostatic ion trapping technique. It is demonstrated that the observed rate of change of ion cyclotron resonance frequency with trapping potential is in excellent agreement with theory, being independent of mass and charge, in the mass range from 1185 to just under 17,000 Da. When a screened cubic trap is used in a ca. 7-T magnetic field, the electrical potential within the trap is effectively reduced, and ion cyclotron resonance frequency shifts as a function of trapping potential are decreased by a factor of 25. There is no evidence of significant improvement in mass resolution with this trap design, except when higher trapping potentials are compared. In a 9.7-V screened trap measurement, a bovine insulin spectrum with an average resolution of 10,000 is obtained. Comparable mass resolution under conventional trapping conditions could not be obtained for trapping potentials greater than 1.2 V. It is evident from this study that use of the screened trap technique could offer advantages for MALDI-Fourier transform mass spectrometry.

Comparative study of photodissociation and surface-induced dissociation by laser desorption Fourier transform mass spectrometry.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.

Energy deposition in iron pentacarbonyl ions undergoing surface-induced dissociation in a Fourier transform mass spectrometer.

Internal energy deposition into iron pentacarbonyl positive ions undergoing surface-induced dissociation (SID) in a Fourier transform mass spectrometer is estimated from the abundances and known critical energies of the product fragment ions. A narrow energy distribution, comparable to that reported in earlier BQ and tandem quadrupole SID studies of the same compound, is observed. As judged by the ratio of fragment ions to incident parent ions observed, SID of iron pentacarbonyl in the 3 T Fourier transform mass spectrometer is more efficient, but results in lower conversion of laboratory to internal energy. This may be a result of the more shallow collision incidence angle employed in the Fourier transform mass spectrometer measurements (a few degrees), which contrasts with the 32-60° collision angles used in the earlier BQ and tandem quadrupole mass spectrometry studies. Collision-induced dissociation with He under single collision conditions is also reported, Not unexpectedly, conversion of kinetic to internal energy was lower than found in a previous Fourier transform mass spectrometer study of the iron pentacarbonyl cation employing argon as collision gas under multiple collision conditions.

Thermal denaturation of alpha-crystallin.

Low molecular weight alpha-crystallin, isolated from cow lenses, exhibit irreversible thermal denaturation in solution at 75 degrees C. The heat of denaturation was obtained from a differential scanning calorimetric endothermic peak. A second run on the denatured alpha-crystallin solution showed that the denaturation is irreversible. Viscosimetric study and ultraviolet absorption spectroscopy also indicated irreversible denaturation.

Water gradients across bovine cornea.

Twenty-six bovine corneas, 30 micron thick, were sectioned perpendicular to the visual axis. Sections, weighing 10-20 mg, were analyzed for total water content by thermogravimetric analysis and for freezable water content by differential scanning calorimetry. The total water content as well as the free (freezable) water content increase, while the bound (nonfreezable) water content decreases in progressing from epithelium to endothelium. These results are correlated with the distribution of proteoglycans and their water sorptive and retentive capacity in bovine cornea.

Topographic correspondence between total and non-freezable water content and the appearance of cataract in human lenses.

Eight intracapsular and 18 extracapsular human surgical specimens were stereophotographed. Each lens was divided into 10 areas, separating those which appeared transparent from those that showed opacities. Samples weighing 6-12 mg from each area were investigated. The total water content was determined by thermogravimetric analysis; the freezable water content was measured by differential scanning calorimetry. The difference between the two provided the non-freezable water content. Graphic presentation illustrates the correspondence of high total water, low non-freezable water content with location of the turbidity in the lens. Pairwise, statistical comparison shows that in intracapsular human surgical specimens the non-freezable water content in the clear areas of both cortex and nucleus was significantly greater than in the opaque areas.

Distribution of the total and non-freezable water in rat lenses.

Twenty Sprague-Dawley rats of ages from 7 days to 29 weeks were studied. The cortical and nuclear samples from rat lenses were investigated by differential scanning calorimetry (freezable water content) and thermogravimetric analysis (total water content). Both cortical and nuclear regions showed a decrease in total water content with aging. Only the nuclear parts of the rat lenses showed a statistically significant increase in the non-freezable portion of the total water with aging. The cortical parts showed a slight but statistically non-significant increase in this parameter. It is concluded that in rat lenses aging is primarily an aggregation process and syneresis does not play a significant role.

Topographic distribution of water in rhesus monkey vitreus.

The topographic distribution of total water content was measured by thermogravimetric analysis in 10 rhesus monkey vitreous bodies. The distribution of the freezable water content in the same vitreous bodies was obtained by differential scanning calorimetry. The nonfreezable water content (bound water) showed a decreasing gradient along the optic axis from lens to fovea; an opposite trend than that found in bovine vitreus. The nonfreezable water content is 25% lower in rhesus monkey than in bovine vitreus which is attributed to the syneresis existent in the rhesus monkey vitreus.