Verification of the DNA predicted amino acid sequence of bacteriophage P22 tail protein by mass spectrometry.

Mass spectrometry was used to verify portions of a proposed amino acid sequence of the bacteriophage P22 tail protein which had been inferred from the DNA base sequence. The exopeptidases dipeptidyl aminopeptidase I and IV and dipeptidyl carboxypeptidase were used to hydrolyse intact protein and fragments generated by cyanogen bromide treatment of the tail protein. After partial purification by high performance liquid chromatography, peptides were identified by gas chromatography/mass spectrometry and fast atom bombardment mass spectrometry. The results indicate that the initiation amino acid, N-formylmethionine, has been removed from the N-terminal of the protein and that the protein ends at the termination codon which is 667 amino acids from the N-terminal residue. Nine regions of the protein from 13 to 42 residues in length were verified. All of the sequences checked were in the same DNA reading frame and corresponded to the proposed sequence.

Protein N-terminal analysis using fast atom bombardment mass spectrometry.

Fast atom bombardment (FAB) mass spectrometry was employed to identify and quantitate dansyl amino acids obtained in the N-terminal analysis of proteins. FAB mass spectra of dansyl amino acids are characterized by intense quasimolecular ions formed by cationization of the molecular species and fragment ions produced by cleavage of the bonds on either side of the sulfanyl group. Investigation of the dansyl amino acid responses using dansyl aminobutyric acid as an internal standard showed that dansyl amino acids can be determined quantitatively at a level of 0.1 nmol. N-terminal residue analysis was performed on a number of proteins to substantiate the technique including bovine serum albumin, pepsinogen, trypsinogen, bromelain, ribonuclease A, and bacteriophage P-22 tail protein.

Synthesis and spectroscopic analysis of modified bile salts.

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.

Fast atom bombardment mass spectrometry of estrogen glucuronides and sulfates.

Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are characterized by abundant ions formed by attachment of a proton, [M+H]+, or of an alkali ion, [M+alkali]+, to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to obtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt.

Periodicities of dinucleotide self-information values in phi X174 DNA.

The natural DNA sequence of bacteriophage, phi X174, when analyzed as a "text" of dinucleotides, is shown to display an easily detectable degree of non-randomness by the distribution of values of dinucleotide self-information along the sequence. Self-information corresponding to occurrences of dinucleotides separated by a single nucleotide is found to be somewhat higher than the values which precede or follow it for every third nucleotide position along the sequence. Consequently autocorrelation coefficients of these values display a strong periodicity and harmonic analysis of the values shows a spike at a value of 3. Self-information autocorrelation periodicity is used as a test of the effect of randomizing portions of the sequence. Any one or two of the three nucleotides in each triplet of the sequence can be chosen at random without losing dinucleotide self-information periodicity except when both the 1st and 3rd nucleotide of all of the triplets in the major phi X174 protein reading frame are randomized. Periodicity is also lost when sequences are generated by randomizing triplets. Autocorrelation and harmonic analysis also indicate other less marked periodic features of dinucleotide self-information values of the nature sequence; non-random features are suggested at periods of 12, 20 and 24 nucleotides.