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Washington University School of Medicine

Applications of Mass Spectrometry in Biophysics

Applications of Mass Spectrometry in Biophysics

One research goal of the Mass Spectrometry Resource is to develop methods to permit study of protein/ligand interactions. To this end, we recently introduced PLIMSTEX (Protein Ligand Interaction by Mass Spectrometry, Titration and H/D Exchange).

PLIMSTEX Figure 1

Example of triation of fatty acid binding protein.

The figure shows an example of a titration of fatty acid binding protein (structure on left above curve) with a fatty acid ligand (potassium oleate) to give a complex (on right) containing the ligand. The solid line is computed from a model that gives the equilibrium constant for the interaction: ((2.6 ± 0.6) x 106 M-1), which agrees with the literature value of 3.0 x 106. We expect application in biophysics and drug discovery, and we are anxious to collaborate with other research groups in extending PLIMSTEX. We also modified PLIMSTEX for the specific case of protein/protein interactions, and we termed this approach Self Interactions by MS, exchange, and titration (SIMSTEX).

Deeper insight can be obtained by digesting the protein and finding those regions where changes in amide exchange occur with ligand binding. Given that mass spectral measurements must be done on a protein for which amide exchange is quenched (pH ~ 2), the digestion uses the enzyme pepsin.

Figure 2

Calcium-binding protein

Difference information comes from the LC/MS and LC/MS/MS analysis of the peptide mixture afforded by the digestion and is displayed as a peptide “protection map,” anexample is shown for the calcium-binding protein, calmodulin. Those portions of the apo (left) and holo (right) that show large decreases in amide exchange upon binding to Ca2+ (yellow balls) are shown in red and orange. For example, the peptide representing the red portion of the central helix of holo (in red) shows a large decrease in exchange in going from apo to holo, consistent with the formation of a tight helix accompanying Ca2+ binding. The blue and black portions exhibit little difference in exchange between the apo and holo forms. We call this approach “High Resolution PLIMSTEX.”

A complementary approach, “Fast Photochemical Oxidation of Proteins” or FPOP is also under development.

FPOP

FPOP uses fast (~ 10 ns) photolysis of H2O2 to produce OH radicals. These radicals “footprint” a protein on the microsecond time scale by reacting rapidly with aromatic, sulfur-containing, and some aliphatic amino acids and oxidize them (usually by substituting an H for OH). Those regions of the protein that are protected by conformation or by binding to a ligand are much less susceptible to oxidation. We learn where the modification (foot printing) has occurred by digesting the protein with trypsin and analyzing the peptide mixture by LC/MS/MS using an identical approach to that used in proteomics. The FPOP procedure is summarized in the diagram where we show the “foot printing” of apomyoglobin in a flow cell by a 17-ns pulse of a 248-nm KrF laser. The radicals are quenched in less than 1 ?s with a chemical scavenger (glutamine, Gln) pre-mixed with the solution. Solvent accessible residues that react with OH are denoted in green on the protein, and when oxidized, denoted in yellow. Each sample “plug” is irradiated once, flows out of the cell, and is protected from repeat irradiation with another “plug” of ~ 20% solution that serves as an irradiation barrier. Two mass spectrometric analyses are done: one of the intact protein to assess extent of oxidation and a second by LC/MS/MS of a tryptic digest to identify amino-acid residues that are oxidized. This footprinting is sufficiently fast that it modifies a protein before it can unfold.

Although the approaches described here continue to be developed, they are available for collaborative research, and interested persons are urged to contact Michael Gross.

Suggested reading:

1. Zhu, Mei M.; Rempel, Don L.; Du, Zhaohui; Gross, Michael L. Quantification of protein-ligand interactions by mass spectrometry, titration, and H/D exchange: PLIMSTEX. J. Am. Chem. Soc. (2003),125, 5252-5253.

2. Hambly, David M.; Gross, Michael L. Laser Flash Photolysis of Hydrogen Peroxide to Oxidize Protein Solvent-Accessible Residues on the Microsecond Timescale. J. Am. Soc. Mass Spectrom. (2005), 16, 2057-2063.

3. Zhu, Mei M.; Chitta, Raghu; Gross, Michael L. PLIMSTEX: a novel mass spectrometric method for the quantification of protein-ligand interactions in solution. Intl. J. Mass Spectrom. (2005), 240, 213-220

4. Chitta, Raghu K.; Rempel, Don L.; Grayson, Michael A.; Remsen, Edward E.; Gross, Michael L.. Application of SIMSTEX to Oligomerization of Insulin Analogs and Mutants. J. Am. Soc. Mass Spectrom. (2006), 17, 1526-1534.

5. Justin B. Sperry, Xiangguo Shi, Don L. Rempel, Yoshifumi Nishimura, Satoko Akashi, and Michael L. Gross, A Mass Spectrometric Approach to the Study of DNA-Binding Proteins: Interaction of Human TRF2 with Telomeric DNA, Biochemistry, 47, 1797-1807 (2008).