The section carries out research in areas of chemistry, biochemistry, and medicine in which mass spectrometry is the primary analytical tool. Current research focuses on the energetics of hydration, protein characterization, and the quantification of endogenous molecules.

Hydration Thermodynamics
Vieira, Gilligan, Yergey
We are determining the energy required for noncovalent bonds between molecules and ions in solution to form and break. The particular bonds of interest are those involved in the interplay of solutes, such as amino acids, small peptides, and lipids, with water. The objective of our thermodynamic investigations is to determine solvation enthalpies and free energies of selected biologically interesting ions. Knowledge of the energetic requirements of these noncovalent bonds, particularly those involving water and biologically significant molecules, is fundamental to understanding molecular interactions and the changes in conformations that are integral to them.

Experimentally, we use the tools of equilibrium gas phase ion-molecule chemistry implemented in a modified electrospray ionization (ESI) source of a single-quadrupole mass filter. In this work, we determine the thermodynamic quantities deltaH(std)298, deltaS(std)298, and deltaG(std)298 by using the approach of equilibrium ion-molecule reaction chemistry. Hydration thermodynamics values are calculated from equilibrium constants measured over a temperature range of 0 to136 C° at ion source water partial pressures ranging from between zero and 100 mtorr. Equilibrium ion intensity measurements were made for at least four hydration states, i.e., zero through three water molecules associated with a core ion, and include at least 60 combinations of water partial pressures and temperatures covering the ranges of experimental variables. An exhaustive study of the equilibrium clustering of one to three waters around n-alkylammonium, CnH(2n+1)NH3+, and the di- and tri-methyl ammonium ions has shown a pattern of behavior consistent with water clusters forming around the charged portion of the ion and being influenced by the nature of the attached hydrophobic groups. Detailed analysis of the entropy values derived from the measurements is consistent with detecting the entropy change associated with the formation of an internal hydrogen bond.

Both the concept of water organizing around a charge site and the formation of internal hydrogen bonds detected by substantial entropy decreases have undergone further exploration with the investigation of the hydration energetics of simple alkyl-diols: 1,2-(OH)2-propane, 1,3-(OH)2-propane, 1,3-(OH)2- butane, and 1,4-(OH)2-butane. The stepwise addition of water to protonated 1,2-(OH)2-propane shows a trend of diminishing exothermicity for each addition. While we were unable to obtain a direct measurement for the addition of the first water, we were able to estimate an upper limit on the exothermicity for the same process based on the water partial pressure and temperature. We conclude that this first hydration step is energetically very favorable. We observed the decreasing trend in energetics for 1,2-(OH)2-propane for the addition of the first two water molecules to 1,3-(OH)2-propane but noted that the decrease was not maintained for the addition of the third water molecule. The addition of the third water to the complex was determined to be energetically more favorable than the addition of the second; in addition, we found a substantial decrease in the entropy for the process. These two observations led us to conclude that the 1,3-(OH)2-propane trihydrate is an energetically and entropically favorable state. The existence of such favorable hydration states is consistent with the observation that the 1,3-diol incorporates itself into several biologically interesting systems and disrupts otherwise stable biomolecular structures. On the other hand, both the butane diols behave in a manner consistent with the 1,2-propane diol.

Recent results from studies of the hydration of several amino acids show great promise for uncovering further structural information about the nature of water clusters and the concept of hydrophobicity. Results from the equilibrium clustering of water with several protonated neutral amino acids (glycine, valine, and leucine) show behavior that is similar to that observed with the alkylammonium ions for the first two water molecules. The addition of the third water, however, is associated with a decrease in both enthalpy and entropy, suggesting the formation of intramolecular hydrogen bonds as seen in the 1,3-propane diol. On the other hand, the results obtained to date in studies of the basic amino acids, arginine and lysine, are almost counterintuitive. That is, while we might expect the energetics of adding the first water to these molecules to be highly favorable, we have determined that it is less energetically favorable than the addition of the first water to the neutral amino acids, suggesting the presence of internal hydrogen bonding before any water clusters are formed.

Protein Characterization
Backlund, Gilligan, Tran, Vieira, Yergey; in collaboration with Blank, Humphrey, Zimmerberg, Crouch, DePamphilis, Epstein, Hinnebusch, Poszgay, Robbins, Rouault, Sackett, Schneerson, Leppla, Levine, Schuck, Alkon, Campbell, Coorssen, Hochberg, Vestal
We conduct research on the mass spectrometric characterization of proteins. We carry out work collaboratively with groups in NICHD as our first priority but also conduct independent investigations. A major aspect of our work is the identification of proteins isolated in biochemical investigations.

For identification of unknown proteins, we use mass spectrometric data to query genomic databases to answer the general question of whether any of the protein sequences present in the databases have expected proteolytic cleavage products with theoretical masses that match the empirically determined masses of the peptides generated from the unknown. Three mass spectrometric approaches are available for this effort: Matrix Assisted Laser Desorption Ionization (MALDI) with Time-of-Flight (TOF) mass analysis; liquid chromatography (LC) followed by electrospray ionization with mass analysis in an instrument capable of using fragmentation reactions to generate peptide sequences (LC-MS/MS); and, most recently, MALDI followed by tandem TOF analysis for the determination of peptide sequences from fragment ion spectra. The last, state-of-the-art technology has become available during the year and has been demonstrated to provide identifications of mixtures of proteins at levels of about 100 fmole. With the current combina-tion of instrumentation, we are confident that, given enough material in a gel band to make 100 fmole available for analysis, we can positively identify a protein that is described in a database.

We are pursuing two development projects designed to improve protein characterization capabilities. First, we have begun addressing the issue of providing sequence information on proteins that are not described in databases because the database either is incomplete or contains errors. We are taking the approach termed complete de novo sequencing of peptides. That approach, which requires detailed interpretation of individual fragmentation mass spectra of peptides, is being implemented in conjunction with software designed and written in our section. To date, we have applied the approach to the sequencing of peptides from a series of standard tryptic digests as well as to tryptic digests of proteins isolated from sea urchin cortical vesicles. Our results have shown that not only does the program identify from a spectrum the correct sequences with the highest-scoring possibility but that the spectra allow Leu residues to be distinguished from Ile. In a related area, we have made substantial progress in the development of a consistent methodology for the identification of site-specific phosphorylation. A combination of collision-induced fragmenta-tion (CID) under LC-MS/MS conditions to identify single phosphorylations and thereby follow neutral phosphate losses with identifica-tion of multiple phosphorylations on single peptides under non–CID conditions with the MALDI TOF/TOF appears to hold considerable promise.

Ratio of Endogenous Cortisone to Cortisol
Li, Yergey; in collaboration with Chrousos, Hochberg, Zoumakis
The purpose of this work is to use mass spectrometry to assess the activity of 11hydroxy-steroid dehydrogenase (HSD) types I and II in both cell culture and clinical studies. HSD-I has recently been shown to contribute to a hormonally dependent obesity when the enzyme is up-regulated in peripheral fatty tissue. The obesity is a consequence of conversion of metabolically inactive cortisone into cortisol through the reduction of a double bond in the steroid by HSD-I. On the other hand, HSD-II is responsible for the reverse conversion. It is hypothesized that increased activity of 11-HSD-I will be recognizable by increased levels of urinary cortisol excretion. As part of a potential therapeutic approach to treating this disorder, an evaluation of 11-HSD-I activity in cultured fibroblasts under conditions of exposure to a variety of candidate agonists is also under way. Experimentally, we have developed an electrospray ionization LC-MS method that is capable of determining the ratio of cortisone to cortisol in both urine and culture media over a 100-fold dynamic range at picomole levels of material injected on column. Preliminary results of these studies have been satisfactory for the purposes of analysis of tissue culture media and for the determination of a suite of urinary steroids.