Principal component analysis of mass spectra of peptides generated from the tryptic digestion of protein mixtures.

Principal component analysis (PCA) has been used to analyse mass spectral peptide profiles obtained from the enzymatic digestion of standard protein mixtures. Scores and loadings plots clearly revealed peptide fragments that differentiated one protein mixture from another. Peptide map search results identified with a high degree of certainty any additional proteins in these mixtures. As a proof-of-concept this methodology was applied to hepatic protein mixtures obtained from rats treated with two hepatotoxic compounds: methapyriline and SB-219994. Liver proteins were extracted, pre-separated by one-dimensional polyacrylamide gel electrophoresis, subjected to tryptic digestion and analysed by mass spectrometry. Two up-regulated proteins, glutathione S-transferase with methapyrilene and peroxisomal bifunctional enzyme with SB-219994, were identified in this manner.

Comparison of LC detection methods in the investigation of non-UV detectable organic impurities in a drug substance.

HPLC Analysis with different detection methods was shown to be essential in the separation and identification of unknown organic impurities in a drug substance. The impurities were found to exhibit very weak or no response to standard ultraviolet (UV) absorption detection. LC-MS, LC-NMR, indirect, refractive index and evaporative light-scattering detection were used to quantify and identify the impurities in this specific case. The drug substance studied was found to be an ideal analyte for demonstrating the advantages and limitations of several chromatographic detection systems for impurity profile analysis.

Comparison of rapid diagnostic methodologies for Chlamydia and gonorrhea in an urban adolescent population: a pilot study.

PURPOSE: To compare a combination DNA probe test which detects both N. gonorrhoeae (GC) and C. trachomatis (CT) to the current culture methodologies among a population of female adolescents at an urban teaching center. In addition, the probe test for CT was compared to a direct immunofluorescence test. METHODS: All sexually active female adolescents between the ages of 13-21 years who sought care at an urban teaching center from June 1991 through November 1991 and who required testing for sexually transmitted diseases (STDs) were recruited for this study. RESULTS: The probe test was demonstrated to be 66.6% sensitive and 94.9% specific when compared to tissue culture for CT and 50% sensitive and 98.2% specific when compared to culture for GC. We found an overall prevalence of 23.5% for CT and 3.5% for GC. CONCLUSIONS: The two rapid diagnostic tests for CT evaluated in this study demonstrated similar sensitivities. However the probe test offers advantages in that it is easier to perform, skill at reading fluorescence is not required, and one specimen yields results for both CT and GC.

Mass balance strategy for protein sequencing. Application to a protein with an extended DNPNNP repeating motif.

The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide a challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.

Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences.

The MN strain of human immunodeficiency virus type 1 was grown in H9 cells, concentrated by centrifugation, and disrupted, and proteins were purified by reversed-phase high-pressure liquid chromatography. Complete amino acid sequences were determined for the mature Gag proteins, showing natural proteolytic cleavage sites and the order of proteins (p17-p24-p2-p7-p1-p6) in the Gag precursors. At least two sequence variants of p24 and eight sequence variants of p17 were detected. The two most abundant variants of p24 and p17 represented at least 50% +/- 5% and 20% +/- 5% of their totals, respectively. These data suggest heterogeneity in the virus population, with 50% of the total virus containing the most abundant forms of p17 and p24 and 20% of the virus containing the second most abundant forms. The Gag precursors of these suggested viruses differ from each other by only 3 amino acid residues but differ from the precursors predicted by the published MN proviral DNA sequence by 10 residues. Electrospray ionization mass spectrometry analysis of the purified p24 forms showed that the measured molecular weight of the protein was 200 +/- 50 atomic mass units greater than the calculated molecular weight. The source of additional mass for the p24 forms was not determined, but the observation is consistent with previous suggestions that the protein is phosphorylated. Greater than 98% of the total recovered p17 was myristylated at the N-terminal glycine residue, and the measured molecular weights (as determined by electrospray ionization mass spectrometry) of the most abundant forms were within 3 atomic mass units of the calculated molecular weights (15,266).

Four-sector tandem mass spectrometric analysis of complex mixtures of phosphatidylcholines present in a human immunodeficiency virus preparation.

A number of phosphatidylcholines have been isolated from an HIV-1/MN preparation by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by fast atom bombardment mass spectrometry (FABMS), FABMS/MS, and FABMS/MS/MS in both positive- and negative-ion modes. Negative-ion FABMS/MS with high-energy collisions was used to identify the length of the acyl groups and the degree of saturation, as well as their position on the glyceride group. FABMS/MS in the positive-ion mode was used to identify the polar head group. Negative-ion FABMS/MS/MS was used to locate positions of double bonds in acyl groups. We find that four-sector tandem mass spectrometry with high-energy collisional activation provides qualitative analysis of viral phosphatidyl lipids in considerable detail, as well as semiquantitative information. Approximate quantitation of the phosphatidylcholine content of the HIV-1/MN preparation by measuring relative peak heights of molecular ions in FABMS reveals an array of phosphatidylcholines consistent with that found in human erythrocytes, indicating the likely source of lipids in the viral membrane to be the host cell membrane.