Mass Spectrometry 1968-1976
Mass spectrometry represents a suite of techniques useful across the entire spectrum of chemical sciences. Originally part of the effort studying isotope effects, studies in mass spectrometry in the Chemistry Division during this period covered several areas: ion-molecule reactions, with an emphasis on energy transfer during ion-molecule reactions, volatilization of large molecules accompanied by gentle ionization techniques, and development of novel mass spectrometric tools, especially those involving mass spectrometry of large biochemical molecules. Because of the importance of learning structural information about large, fragile biomolecules, the latter two topics are closely related.
Whether the ions to be analyzed in a mass spectrometer are created by electron impact, photoionization, or by chemical reaction, reactions between molecular ions and neutral molecules can modify the result. Therefore, it is important to understand the mechanisms of ion-molecule reactions in order to interpret mass spectra correctly.
When atomic and molecular ions impact neutral molecules, excited product ions can be generated which have sufficient internal energy to decompose. Generally, several competing chemical and energy-transfer decomposition channels are available to the excited ions, and analysis of the resulting products under varying conditions reveals information about ion-molecule reaction mechanisms.
Hydrocarbons such as cyclopropane and propylene were subjected to mass-selected rare gas, hydrogen, deuterium and mixed rare-gas hydrogen and deuterium ion collisions. Analysis of the energy and identities of the products, along with the known energy levels of the reactants, served to reveal details of the reaction mechanisms. For instance, the data suggested that processes which could have been simple hydride transfers actually took place as H2 molecular elimination from an excited ion.1
1. "Charge Transfer and Proton Transfer in Polyatomic Ion-Molecule Systems" J.J. Leventhal and L. Friedman, J. Chem. Phys. 48 1559 (1968).
Mass Spectrometry of Large Molecules: Gentle Ionization Techniques
Generally speaking, molecules containing large numbers of atoms have lower vapor pressures than molecules with fewer atoms. Since mass spectrometry at this time depended on volatilizing measurable concentrations of neutral molecules prior to ionization, it was thought that mass spectrometry would only be limited to studies of relatively small molecules. An additional complication is that even if large molecules could be volatilized at high temperatures they would have more internal energy and would fragment more extensively during ionization. A study was undertaken to measure the amount of fragmentation of a comparatively large biomolecule as a function of the ionization energy. It was found with a derivatized tripeptide that the yield of the protonated parent ion was half the total ion yield when gentle protonation was done with a beam of D9O4+. When less gentle protonation was accomplished with H3O+, the parent ion was only 1% of total ion yield.2 Thus even though the large molecule had vaporized intact, an energetic ionization process could fragment the biomolecule into small fragments, leading one to think the molecule had fragmented during evaporation. Achieving control of fragmentation during the ionization process separate from fragmentation during vaporization then allowed studies of volatility enhancement of large molecules.
2. “Solvated Proton Mass Spectra of a Tripeptide Derivative” R.J. Beuhler, L.J. Greene and L. Friedman, J. Am. Chem. Soc. 93 4307 (1971).
Mass Spectrometry of Large Molecules: Volatility Enhancement Techniques
During this period experiments were performed in the Chemistry Division establishing methods for volatilizing biomolecules without decomposing them. Methods such as derivatization to increase vapor pressure and to suppress decomposition were investigated. These methods were less satisfactory since the nature of the molecule being examined was changed, and side reactions yielded unwanted products.
A study of the evaporation of synthetic peptides as a function of their binding energy to surfaces was undertaken. Various surfaces including platinum, glass, carbon, copper and Teflon® were used and sample size and dispersion were varied. When thyrotropin releasing hormone (TRH, mass=362 mass units) was sublimed into a mass spectrometer from the surface of a hot platinum filament, no parent or quasi-parent (mass+proton) ions were observed with gentle ionization. The parent molecule was completely decomposed at high temperature, and concentrations of suitable fragment ions were low. However, when small (5 mg) samples of TRH were deposited from methanol solution on carbon, Teflon®, or glass surfaces, and then were sublimed at relatively modest (<300°C) temperatures, sufficient concentrations of quasi-parent and fragment ions were obtained to permit determination of the amino acid sequence. The lower binding energy of molecules to these surfaces permits volatilization under milder conditions, so that molecules were much less fragmented. The lower activation energy of sublimation from such surfaces also increased the rate of desorption, enhancing the concentration of sample in the gas phase.3
3. "Volatility Enhancement of Thyrotropin Releasing Hormone for Mass Spectrometric Studies" R.J. Beuhler, E. Flanigan, L.J. Greene and L. Friedman, Biochem. Biophys. Res. Commun. 46 1082 (1972).
Mass Spectrometry of Large Molecules: Rapid Heating Techniques
During the course of the volatility enhancement experiments for large molecules a study of the activation energies for evaporation and decomposition of large molecules was undertaken. In general it was found that the evaporation of large molecules had a larger activation energy than the activation energy for decomposition on the surface. This meant that at lower temperatures decomposition would predominate, but if the sample could be heated rapidly then it would evaporate intact. This lead to the technique of rapidly heating the sample from inert surfaces to increase the amount of sample vaporized compared to the amount decomposed. It was found that underivatized two-to-five amino-acid peptides containing arginine could be vaporized intact. Gentle ionization caused fragmentation which proceeded via breakage of peptide linkages and elimination of small, neutral molecules. Fragmentation was sufficiently controlled that amino acid sequences could be determined. Even the nine-amino-acid peptide bradykinin yielded spectra structure correlations sufficient to derive a unique amino acid sequence.4
4. "Proton Transfer Mass Spectrometry of Peptides. A Rapid Heating Technique for Underivatized Peptides Containing Arginine" R.J. Beuhler, E. Flanigan, L.J. Greene and L. Friedman, J. Am. Chem. Soc. 96 3990 (1974).
These pioneering experiments utilizing rapid sample heating accompanied by gentle ionization helped lead the way toward mass spectrometric analysis of even larger biomolecules in the subsequent decades.
Last Modified: February 9, 2016