Ammonia laser desorption/chemical ionization with ammonium bromide: Fourier transform ion cyclotron resonance mass spectrometry of aromatic hydrocarbons.

Laser desorption/ionization combined with Fourier transform ion cylcotron resonance mass spectrometry (LD/FT/ICR/MS) is a proven technique for the analysis of nonvolatile materials. Unfortunately, LD tends to produce a large excess of neutral species compared to ions. Laser desorption followed by chemical ionization (LD/CI) by use of a reagent gas is a seIective and sensitive means of control in the analysis of nonvolatile compounds. In this article we demonstrate the technique of ammonia LD/CI by addition of a small amount of ammonium bromide (NH4Br) to an involatile sample, i.e., the ammonium salt is used in place of ammonia reagent gas. For various aromatic hydrocarbons, abundant (M + H)(+) ions are produced as a result of CI A primary advantage of this method in FT/ICR/MS is that selective LD/CI experiments may be conducted at low pressure as in pulsed valve CI (but without the need for pulsed valve operation), thereby providing the potential for obtaining high-resolution FT/ICR mass spectra.

High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus.

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.

Selective parent ion axialization for improved efficiency of collision-induced dissociation in laser desorption-ionization fourier transform ion cyclotron resonance mass spectrometry.

We have systematically established the excitation frequency, amplitude, duration, and buffer gas pressure for optimal axialization efficiency and mass selectivity of quadrupolar excitation-collisional cooling for isolation of parent ions for collision-induced dissociation in Fourier transform ion cyclotron resonance mass spectrometry. For example, at high quadrupolar excitation amplitude, ion axialization efficiency and selectivity are optimal when the applied quadrupolar excitation frequency is lower than the unperturbed ion cyclotron frequency by up to several hundred hertz. Moreover, at high buffer gas pressure (10(-6) Torr), quadrupolar excitation duration can be quite short because of efficient collisional cooling of the cyclotron motion produced by magnetron-to-cyclotron conversion. Efficiency, detected signal magnitude, and mass resolving power for collision-induced dissociation (CID) product ions are significantly enhanced by prior parent ion axialization. With this method, we use argon CID to show that C 94 (+) (m/z 1128) formed by Nd:YAG laser desorption-ionization behaves as a closed-cage structure.

Using solution phase hydrogen/deuterium (H/D) exchange to determine the origin of non-covalent complexes observed by electrospray ionization mass spectrometry: in solution or in vacuo?

Electrospray ionization (ESI) is a soft ionization technique that is able to transfer intact ions, as well as solution phase non-covalent complexes into the gas phase. With small molecules that have a high tendency to form hydrogen bonds, the observation of non-covalent complexes by ESI-MS can be the result of a non-specific interaction, due to the nature of the electrospray process. Special precautions and additional steps should be performed to identify the origin of the complexes observed with ESI-MS, and we have utilized solution phase hydrogen/deuterium (H/D) exchange as a method to determine the specificity of the complexes. By comparing the average number of exchanges for the monomer subunits to the average number of exchanges for the complex, one can distinguish if a specific complex is formed in solution. In this paper we have investigated non-covalent complexes of some common chemotherapy agents: paclitaxel, doxorubicin, and etoposide by ESI-MS. By using the solution phase H/D exchange, we were able to identify several specific drug-drug complexes. Thus, solution phase H/D exchange combined with ESI-MS provides for a convenient method in ascertaining the specificity of non-covalent complexes as being formed in solution or in vacuo.

Broadband quadrupolar axialization of large multiply charged ions to enhance measurement and minimize conformational restrictions.

For high-resolution Fourier transform mass spectrometry of electrosprayed proteins, the signal-to-noise ratio of measuring nozzle-skimmer fragment ions can be improved substantially by their broadband quadrupolar axialization (QA), even without increasing their concentration in the ion cyclotron resonance cell. Axialization of the product ions makes possible larger, more concentric ion orbits for measurements. QA allowed the identification of new sequence-indicative product ions from a 29 kDa protein. However, QA of large molecular ions gives little increase in signal, consistent with original trapping near the cell axis. By recentering of ions undergoing ion-molecule reactions, these can be carried out at much higher kinetic energy and pressures; for cytochrome c this increases the achievable H-D exchange by 40%, corresponding to exchange at all the active sites of its completely denatured conformer.

PET study of the localization and laterality of lingual somatosensory processing in humans.

Regional cerebral blood flow (rCBF) was measured using positron emission tomography (PET) during four tasks in right-handed volunteers with eyes closed: resting, protruding the tongue, stroking the left side of the protruding tongue, and stroking the right side of the protruding tongue. The primary somatosensory tongue representation (S1) mapped to the contralateral central sulcus (Brodmann (BA) 3/4) at approximately 28 mm above the intercommissural plane. Of note, stimulation of the left side of the tongue produced also an ipsilateral S1 response. Analysis of variance (ANOVA) of rCBF at S1 across all four conditions yielded only a significant effect for tongue stimulation, with no effect of laterality; the usually large asymmetries (contralateral >> ipsilateral) in S1 did not surface. We hypothesize that this atypical activation pattern arises from the tongue's specialization for language.

Retrospective analysis of a novel method of transscleral suture fixation for posterior-chamber intraocular lens implantation in the absence of capsular support.

PURPOSE: To determine the safety and efficacy of an alternative method for transscleral fixation of a secondary posterior-chamber intraocular lens (pcIOL) during penetrating keratoplasty. METHODS: Eighty-nine eyes that underwent secondary pcIOL implantation by using a modified transscleral suture-fixation technique during penetrating keratoplasty were retrospectively evaluated. The surgical technique used suture fixation to the surface of the sclera 5 mm posterior to the limbus, with the knot buried beneath Tenon's capsule and conjunctiva. Patient records were reviewed for postoperative complications, including suture erosion, pcIOL subluxation, vitreous hemorrhage, and retinal detachment. Mean follow-up was 24.4 months, with a range of 4-68 months. RESULTS: All eyes had successful fixation of their pcIOL immediately after surgery. Three (3.3%) eyes had graft failure. Six (6.7%) of 89 eyes showed evidence of suture erosion or partial exposure. Postoperative suture breakage occurred in two (2.2%) eyes. Posterior-segment complications included retinal detachment in one (1.1%) eye, vitreous hemorrhage in one (1.1%) eye, and limited choroidal hemorrhage in two (2.2%) eyes. Median visual acuity at 1-year follow-up was 20/70 (range, 20/25 to light perception). CONCLUSION: This transscleral fixation technique provides a straightforward alternative to previously described techniques. Suture erosion, IOL dislocation, and posterior-segment complications occurred at relatively low rates compared with other pcIOL implantation techniques.

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry.

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.

Quantification of biomolecules by external electrospray ionization Fourier transform mass spectrometry.

Fourier transform mass spectrometry (FTMS) is well-known for its capabilities in structural characterization of molecules. Recent developments in radio frequency excitation, linearized trapping, and accumulation of ions generated from external sources have improved the potential of FTMS for quantitative analysis. Here, a commercial external electrospray ionization FTMS, employing a linearized ion trap (the Infinity Cell) and an ion accumulation procedure in which ions are deflected off-axis and injected into the trap, is evaluated as an analytical method for quantifying amino acids, peptides, and proteins. Linear response over approximately 2-3 orders of magnitude is observed for singly-charged ions with low coefficients of variation (generally < 10%), and the calibration curves generated can be used to quantify structurally similar analytes with < 4% relative error, as shown here for quantification of leucine enkephalin from curves generated by methionine enkephalin. Similar precision is obtained for multiply-charged lysozyme, but over only 1.5 orders of magnitude. Some m/z discrimination is observed as a function of trap accumulation potential for a two-component cytochrome c/lysozyme mixture. The results are promising because they suggest that quantification using liquid chromatography coupled to electrospray FTMS is possible.

Thin gold film-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry of biomolecules.

A new thin gold film-assisted (TGFA) laser desorption/ionization (LDI) technique has been combined with Fourier transform ion cyclotron resonance (FT-ICR) high-resolution mass analysis. A thermally labile organic sample is coated onto a thin gold film deposited on a glass plate and desorbed and ionized by Nd:YAG laser irradiation. The wavelength for maximum light absorption may be tuned by varying the thickness of the metal film; e.g., a 10-nm-thick gold film absorbs maximally near the Nd:YAG fundamental wavelength (1064 nm). A key advantage of the method is the stability of the gold film, which facilitates deposition of samples from any of a variety of solvent systems. Coupled with recently introduced quadrupolar excitation and collisional cooling for ion axialization, TGFA-LDI FT-ICR mass spectra provide high sensitivity (e.g., 100-fmol single-shot detection limit and mass resolving power, m/delta m approximately 113,000, for (M+K)+ ions from gramicidin S at m/z 1180).

Electrospray ionization fourier transform mass spectrometry quantification of enkephalin using an internal standard.

Fourier transform mass spectrometry (FTMS), long-known for its capabilities in structural characterization of molecules, is an emerging tool in quantification, and quantification methods using external and internal standards with electrospray ionization (ESI) FTMS have recently been demonstrated. Here, commercial ESI-FTMS is used to quantify the opioid pentapeptide methionine enkephalin using an internal standard. Linear working curves over three orders of magnitude are obtained using the internal standard, an improvement of one order of magnitude over the previous external standard ESI-FTMS quantification method for enkephalins. Low coefficients of variation (generally <6%) are observed, and inter-day and intra-day assays are compared and found to possess similar linearity and precision. The high mass accuracy advantage of FTMS can be exploited to give molecular specificity. Efforts to improve mass accuracy using internal mass calibration generally provide mass accuracies within 2.5 ppm.

Enhanced mass resolving power, sensitivity, and selectivity in laser desorption Fourier transform ion cyclotron resonance mass spectrometry by ion axialization and cooling.

Ion cooling and axialization produced by azimuthal quadrupolar excitation in the presence of ion-neutral collisions are applied to laser desorption Fourier transform ion cyclotron resonance mass spectrometry (LD/FT/ICR-MS). With this technique, the large kinetic and internal energies of ions generated by laser desorption processes can be cooled effectively by collisions of ions with neutral argon atoms (at > 5 x 10(-7) Torr). After sufficient cooling in the source compartment of a dual ion trap, the axialized ions may be transferred to the analyzer compartment for detection at much lower pressure (and thus much higher mass resolving power). Enhancements in both FT/ICR mass resolving power and sensitivity are observed; moreover, ion isolation with high selectivity at high pressure is also demonstrated.

Creatine kinase: essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry.

Phenylglyoxal is an arginine-specific reagent that inactivates creatine kinase (CK). Previous results suggest that modification of the dimeric enzyme at a single arginine residue per subunit causes complete inactivation accompanied by the loss of nucleotide binding; the actual site of modification was not identified. Here, high-resolution tandem mass spectrometry (MS/MS) was used to identify three phenylglyoxal-modified Arg residues in monomeric rabbit muscle CK. Electrospray ionizaton Fourier-transform MS of the phenylglyoxal-modified CK that had lost approximately 80% activity identified three species: unmodified, once-modified (+116 Da), and twice-modified (+232 Da) enzyme in a ratio of approximately 1:4:1. MS/MS restricts the derivatized sites to P122-P212 and P283-V332, whereas MS of Lys-C digestions revealed two modified peptides, A266-K297 and G116-K137. The only Arg in A266-K297 is Arg-291 (invariant), whereas MS/MS of modified G116-K137 shows that two of the three sites Arg-129, Arg-131, or Arg-134 (all invariant) can contain the modification. The recently reported x-ray crystal structure for the octameric chicken mitochondrial CK indicates that its nucleotide triphosphate-binding site indeed contains the equivalent of R291, R129, and R131 reported here to be at the active site of rabbit muscle CK.

Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.

Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.

Direct sequence data from heterogeneous creatine kinase (43 kDa) by high-resolution tandem mass spectrometry.

Isoelectric focusing separation of recombinant rabbit muscle creatine kinase (CK) and its 282Cys-->282Ser mutant shows the presence of three and two isoforms, respectively, that exhibit equivalent enzymatic activity. Electrospray ionization coupled with Fourier-transform mass spectrometry (10(5) resolving power) of both CKs indicates that their major components are within +/- 2 Da of the M(r) value predicted from the cDNA sequences of these mixtures. Dissociation of (M + nH)n+ gives no evidence that the components of either CK are isomers; the masses of the 51 fragment ions correlate completely (+/- 1 Da) with the values predicted from the cDNA sequence and confirm the identities of 21 of the 380 amino acids and the 282Cys-->282Ser replacement in the mutant. The results are consistent with one or two steps of post-translational amidation/deamidation (NH2-->OH, 16 Da-->17 Da), each of which would produce only a 1 Da difference in M(r), with the fragment masses indicating that at least one modification occurs between residues 212 and 282.

Surface-induced dissociation of multiply-protonated proteins.

A novel surface design compatible with the open cell geometry allows nonglancing angle collisions of selected ions stored in a Fourier transform mass spectrometer. Dissociation efficiencies of 36%, 22%, and 14% are achieved for gramicidin S, melittin, and carbonic anhydrase (29 kDa), respectively. Ion neutralization by the surface, which is highly competitive for many singly-charged ions, is minimal, and dissociation products of hypervalent neutral species are not detected. Instead, the spectra are similar to those from collisionally activated and infrared multiphoton dissociation; the fragmentation pathways are relatively independent of the method of energy deposition. For carbonic anhydrase, however, the single event excitation inherent to surface-induced dissociation appears to minimize secondary fragmentation, a critical advantage for tandem mass spectrometry of such large ions. Electrically floating the open cell below ground greatly enhances the collection efficiency.

Gas-phase folding and unfolding of cytochrome c cations.

Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D2O, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.