Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon ␣-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.
The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.
The Akabori reaction, devised in 1952 for the identification of C-terminus amino acids, involves the heating of a linear peptide in the presence of anhydrous hydrazine in a sealed tube for several hours. We report here a modified Akabori reaction that rapidly identifies the C-terminus amino acid in a polypeptide including its amino acid sequence information at both the C-terminus and the N-terminus. This modified methodology demonstrates the fundamentals of microwave chemistry applied to bioanalytical problems. In this modified process, hydrazinolysis has been accelerated by the application of microwave irradiation. In our reaction, the linear peptide and hydrazine solution, contained in a loosely covered conical flask, was exposed to a few minutes of irradiation using an unmodified domestic microwave oven. While the classical Akabori reaction required several hours, the microwave assisted reaction takes just minutes. If dimethyl sulfoxide is added to dilute the reaction mixture, the process is retarded enough to allow aliquots of the reaction mixture to be drawn every few minutes over a period of about an hour in order to study the progress of hydrazinolysis. Reaction products were monitored by mass spectrometry-primarily FAB-MS. In addition to providing sequence information, the microwave enhanced Akabori reaction quickly detects the presence of arginine (Arg) by converting each Arg to ornithine (Orn). Furthermore, certain amino acids, containing -SH, CO 2 H, and CONH 2 groups in their side chain, are susceptible to modification by hydrazine, thereby, providing rapid confirmation of the presence of these amino acid residues. In these preliminary studies, the following oligopeptides were analyzed to demonstrate the effectiveness of our approach; the dipeptide (Trp-Phe), the tripeptide (Tyr-Gly-Gly), the tetrapeptide (Pro-Phe-Gly-Lys), the heptapeptide (Ala-Pro-Arg-Leu-ArgPhe-Tyr), and a N-terminal blocked tripeptide (N-acetyl-Met-Leu-Phe). (J Am Soc Mass Spectrom 2002, 13, 839 -850)
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