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Ion spectroscopy

cyto_all_G-fit2 Ion "action" spectroscopy combines the sensitivity and separation capabilities of mass spectrometry with the structural insights from spectroscopy. The electronic or vibrational spectra of ions are measured by mass selecting an ion of interest via mass spectrometric means, and then irradiating the ions with a bright tunable light source, such as a laser. Resonant absorption will cause an increase in the internal energy of the ion, which can then lead to dissociation (i.e., a change in mass, as measured by the mass spectrometer). Note that in this "action" or "consequence" scheme, it is not the absorption of the light that is monitored, but rather its consequence, namely dissociation. Ion spectroscopy has a rich history in fundamental gas-phase science, as discrete complexes can be selected (by mass), and their inherent structure (in the absence of solvent or other effects) can be probed. A good example is shown in the Figure to the right, where the secondary structure of a peptide can be confirmed based on diagnostic NH and OH stretches [1].

1. Bioanalytical applications

A) Isobaric, isomeric differentiation

cyto_all_G-fit2 Despite the promise of spectroscopy for obtaining structural information on (bio)molecules, ion spectroscopy is far from being a routine bioanalytical tool. Our group aims to change this by developing methodologies for molecular systems where current approaches are lacking. The Figure to the left shows IR spectra of isobaric peptides that are sulfated or phosphorylated [2]. sYG denotes sulfated, pYG denotes phosphorylated, and YG denotes the unmodified peptide. Various diagnostic modes can be discerned, such as the sulfate OH stretch, phosphate OH stretch, alcohol OH stretch, and carboxylic acid OH stretch. This underlines the ability of IR spectroscopy in identifying chemical groups in molecules based on diagnostic vibrations.

cyto_all_G-fit2 The Figure to the right illustrates that sulfated peptides selectively dissociate at 3590 cm-1, whereas phosphorylated peptides dissociate at 3670 cm-1. By monitoring these selected wavelengths, sulfated and phosphorylated peptides could thus be screened from a mixture of enymatic peptides. These band positions are found to be reproducible in larger peptides also. The significant difference in the IR band positions of sulfate and OH modes contrast with the very small differences in mass between both of these isobaric species.

cyto_all_G-fit2 Carbohydrates (a.k.a. sugars, saccharides) are among the most extreme examples of isomeric heterogeneity in molecules. The structures merely differ in their building blocks, linkage positions, and anomericities of their glycosidic bonds. The Figure to the right shows how trisaccharides can be fragmented to disaccharide units, which can then be structurally interrogated with tunable IR radiation [3]. The photodissociation mass channels of the α1-4 and α1-6 are plotted as heat maps (blue = low intensity, red = high intensity), showing distinct differences between these isomeric C2 fragments.

These projects are funded by the National Science Foundation under grant CHE-1403262.

B) Structural characterization of unknown metabolites

cyto_all_G-fit2 Current MS-based methodologies to identify metabolites involve liquid chromatography (LC) for separation, followed by MS/MS fragmentation. The main bottleneck in identification lies in the limited number of standards for which reference MS/MS spectra are available. The application of IR ion spectroscopy to metabolite identification introduces another dimension of information. Some of this information can be relatively straightforard to interpret, such as the presence (or absence) of various chemical groups based on diagnostic band positions. Nonetheless, there are in principle a very large number of molecules that have a particular mass [4], making it all the more essential that detailed structural information on unknowns is obtained. The Figure to the left illustrates why IR ion spectroscopy has the potential to be game changer in this respect [5]. For a metabolic pathway with known metabolites (a-d), it is not apparent from the mass of an unknown (m/z 286.193) that it is related to this cycle. Conversely, its IR spectrum is likely to have high spectral similarity to the IR spectra of the known metabolites a-d. As the IR spectral similarity strongly correlates with the molecular similarity, a comparison of the IR spectrum of an unknown to reference IR spectra of standards should readily allow a chemical classification of the unknown. With the additional mass information, this provides a strategy for formulating all of the putative candidate structures for this unknown. This strategy relies on establishing an IR database of standard metabolites.

Some of the challenges in making ion spectroscopy a routine bioanalytical tool lie in the technical difficulties in coupling tunable light sources with mass spectrometry instrumentation. This is particularly relevant because the highest spectral resolution (and thus highest analytical specificity) is attained at temperatures close to absolute zero. We are therefore developing cryogenic instrumentation, where the ions can be cooled in cryogenic traps (see Figure to the right).

This project is funded by the National Institutes of Health under grant number R01GM110077.

2. Fundamental studies

A) Biomolecules in the gas phase

cyto_all_G-fit2 Fundamental studies in our lab have aimed to gain insights on processes/methodologies that underpin bioanalytical MS. We have shown that labile post-translational modifications, such as sulfations, can migrate from one site in the peptide to another site [6]. Such migrations may lead to incorrect identification of the sulfation site in the peptide in sequencing experiments.

In a recent paper, we have shown that protonation sites that are favorable in solution may be conserved in the gas phase in electrospray ionization (ESI) [7]. In the Figure to the right, para-aminobenzoic acid (PABA) exists as the amino-protonated (i.e., NH3+) in solution. When using protic solvents in a "hot" ESI source, the structure isomerizes to the gas-phase favorable carboxylic acid-protonated form (i.e., CO2H2+). Conversely, when spraying from aprotic solvents in a "cold" source, the solution favored amino-protonated structure is retained. Retaining the less favorable structure in the gas phase is also referred to as kinetic trapping, which may make the identification of analytes based on reference IR spectra more challenging.

B) Biomolecules on interstellar dust particles

cyto_all_G-fit2 Using matrix isolation instrumentation, we are simulating conditions on interstellar dust particles to elucidate the reaction mechanisms that can give rise to biomolecules in the interstellar medium. Of particular interest is the simplest amino acid glycine, which may arise radical dissociation products from methylamine and formic acid. The identities and concentrations of the various reagents and products are verified with FTIR spectroscopy in a matrix isolation setup [8]. The radical reaction products are generated via UV photolysis.