Gary R. Kinsel

Profile and flight time analysis of bovine insulin clusters as a probe of matrix-assisted laser desorption/ionization ion formation dynamics

Detailed ion signal profile and flight time analyses were performed on the time-of-flight signals obtained for bovine insulin cluster ions produced by matrix-assisted laser desorption/ionization (MALDI). Profile analyses of the ion signals strongly suggest that the signals are made up of a composite of two separate components and analysis of the flight times of the two components suggests that the ions are formed with different dynamics. The formation dynamics of one component of the ions is best described as prompt ionization to yield ions with essentially mass-independent total energies. The formation dynamics of the second component of the ions is best described as delayed gas-phase ionization of entrained material moving at a constant velocity. This model is supported by several ancillary experiments and suggests new insights into the mechanism of MALDI ion formation.

Investigation of the mechanism of intracluster proton transfer from sinapinic acid to biomolecular analytes

Experiments have been performed to elucidate the mechanism of proton transfer in ternary clusters containing the matrix-assisted laser-desorption ionization (MALDI) matrix sinapinic acid, nonchromophoric analytes (proline, methionine, and prolylmethionine), and argon. To investigate the mechanism of intracluster proton transfer, ionizing laser power studies were performed at 266 and 355 nm. Baseline studies show that two photons are required at both wavelengths for the formation of sinapinic acid radical cations from sinapinic acid/argon clusters. Studies of the ternary sinapinic acid/biomolecule/argon clusters show that, in all cases, the photon dependence for protonation of the biomolecule is the same as that for formation of the sinapinic acid radical cation. Furthermore, the slopes of the power plots are generally between 1.5 and 2.0, consistent with a two photon ionization process. No evidence of negative ion formation is detected in the negative ion mass spectra. The combined results are consistent with a mechanism of biomolecular intracluster protonation via proton transfer from the photoionized sinapinic acid radical cation. Wavelength dependent trends in matrix and analyte fragment ion formation in conventional MALDI mass spectra and the cluster proton transfer mass spectra were noted. The possible contribution of cluster proton transfer to the analyte protonation mechanism in conventional MALDI is discussed.

Investigation of the dynamics of matrix‐assisted laser desorption/ionization ion formation using an electrostatic analyzer/time‐of‐flight mass spectrometer

A hybrid electrostatic analyzer/time-of-flight mass spectrometer was used to examine the matrix-assisted laser desorption/ionization (MALDI) ion kinetic energy distributions for Na + , protonated 7-hydroxy-4-methylcoumarin matrix ions and protonated bradykinin under a variety of source region accelerating electric field conditions. Broad kinetic energy distributions are observed for the three targeted ions with the ions having both positive and negative kinetic energies relative to the full acceleration potential. The positive kinetic energy ions are shown to have flight times in good agreement with flight times calculated from first principles while the flight times of the negative kinetic energy ions diverge positively from the calculated values. Analysis of the flight time shifts for the negative kinetic energy ions allows an entrainment velocity of ∼550 m s -1 for the material to be calculated. A composite picture of the dynamics of MALDI ion formation is presented which combines promptly formed protonated matrix and analyte ions with ions formed/accelerated at delayed times in an expanding, constant velocity plume of laser desorbed material.

Calixarene amino acids; building blocks for calixarene peptides and peptide-dendrimers

Abstract A modular strategy towards receptor macromolecules is presented, which combines synthetically diverse peptide synthesis with highly functional calixarene chemistry. The design and synthesis of calix[4]arene amino acids 1a – f , calix-lysines, is described, which were used as construction blocks to assemble nanoscale, multivalent entities—calix–peptides 2 and calix–peptide-dendrimers 3 .

Photodissociation of High Molecular Weight Peptides and Proteins in a Two-Stage Linear Time-Of-Flight Mass Spectrometer

A two-stage linear time-of-flight mass spectrometer is used to investigate the requirements for performance of laser photodissociation of peptide and protein ions. Results are presented that demonstrate that desorption and dissociation laser pulses can be synchronized to irradiate ions that travel at high velocities down the drift tube of a time-of-flight mass spectrometer. For example, 193-nm photodissociation of bovine insulin and doubly charged lysozyme is demonstrated, and laser power studies suggest that dissociation is initiated by the absorption of a single 193-nm photon. These results are encouraging because they suggest that laser photodissociation of high molecular weight proteins can lead to fragmentation on time scales compatible with time-of-flight mass spectrometry.

Effects of heavy-atom substituents on matrices used for matrix-assisted laser desorption-ionization mass spectrometry

The data reported show that heavy-atom substitution on matrices that are used for matrix-assisted laser desorption-ionization mass spectrometry enhance [M + H]+ ion yields of both Leu—enkephalin and vitamin B12. Heavy-atom substitution alters the excited state relaxation dynamics of the matrices as indicated by measured decreases in the measured luminescence (fluorescence and/or phosphorescence) observed for heavy atoms that contain matrices relative to the nonheavy-atom-substituted matrices. The results presented are consistent with the direct involvement of an electronically excited state of the matrix in the formation of analyte [M + H]+ ions.

Quantitative determination of the peptide retention of polymeric substrates using matrix-assisted laser desorption/ionization mass spectrometry

Polymer surface-peptide binding interactions have been shown previously to lead to reductions in peptide matrix assisted laser desorption/ionization (MALDI) ion signals. In previous studies, increases in surface-peptide binding were characterized by the increases in both the initially adsorbed and retained quantities of 125I-radiolabeled peptides. The present studies establish a specific correlation between the peptide retention properties of the polymer surface and the reduction in the peptide MALDI ion signal. This correlation is demonstrated by obtaining MALDI mass spectra of angiotensin I applied to various polymer surfaces having a range of peptide adsorption and retention properties. In addition, the use of a MALDI based method of standard additions is shown to allow the quantitation of the polymer surface—peptide retention affinity for angiotensin I and porcine insulin. The MALDI standard additions method for measurement of surface-peptide retention affinities offers a number of significant advantages over conventional radiolabeled peptide binding methods and promises to be a valuable tool for the determination of this important biomaterial characteristic.

Design and calibration of an electrostatic energy analyzer-time-of-flight mass spectrometer for measurement of laser-desorbed ion kinetic energies.

The design of a hybrid electrostatic energy analyzer-time-of-flight mass spectrometer for measurement of ion kinetic energies produced by laser desorption ionization is presented. The need for experimental evaluation of the calibration and performance of the instrument is discussed and a novel laser multiphoton ionization technique, which allows experimental calibration of the energy bandpass of the electrostatic energy analyzer, is described. Laser multiphoton ionization at varying electric field strengths also allows the effects of electric field distortions on energy resolution of the instrument to be probed. Measurement of the translational energies of ions produced by 266-nm laser desorption ionization at 48 mJ/cm2 of material adsorbed to a stainless steel probe by using this instrument also is presented. Ion translational energies of +19±5, +10±5, and +10±5 eV are found for adsorbed Na+, K+, and m-xylene M+, respectively.

Mechanistic investigation of intracluster proton transfer from p-hydroxybenzoic acid to peptides

The mechanism of photoinitiated intracluster proton transfer from the laser desorbed proton donor, p-hydroxybenzoic acid, to various co-desorbed, non-chromophoric peptide proton acceptors has been investigated. Both laser power and wavelength-dependent data are presented which suggest a mechanism of cluster proton transfer initiated by two-photon ionization of the p-hydroxybenzoic acid. The mass spectra resulting from the photoinitiated cluster proton transfer reaction are shown to contain ion signals from both the protonated peptide parent molecule and peptide fragments. Examination of the specific fragment ions observed and fragment ions produced from a series of complementary dipeptide pairs suggests that the fragment ions result from peptide activation during the cluster proton transfer reaction. Furthermore, N-terminal protonation of the peptide proton acceptors is suggested in the absence of arginine or histidine residues.

MALDI-MS as a monitor of the purification and folding of synthetic eclosion hormone.

Analogues of the small protein Manduca sexta eclosion hormone (62 amino acids) were synthesized by Fmoc solid-phase methodology. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze the products of the syntheses and this information was used to design an efficient purification scheme. MALDI-MS was used to monitor the target products through purification and it was also used to monitor folding of the purified materials. The folded EH analogues were shown to be biologically active proteins with an in vivo bioassay using pharate adult moths, Heliothis virescens.