Tandem mass spectrometry methods for definitive protein identification in proteomics research.

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure.

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Identification of glyceraldehyde-3-phosphate dehydrogenase as a cellular protein that binds to the hepatitis B virus posttranscriptional regulatory element.

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA cis-element that is required for high-level expression of viral surface gene transcripts and appears to function by activating mRNA export to the cytoplasm. We have previously shown that multiple fragments of the PRE bind to two cellular proteins of approximately 35 and 55 kDa in molecular mass and that this binding correlates with function. By a combination of column chromatographic techniques and SDS-polyacrylamide gel electrophoresis, we have been able to purify the smaller protein. Amino-terminal sequencing of the purified protein shows identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an RNA-binding glycolytic enzyme that has been implicated in the export of tRNA. Immunoprecipitation analysis reveals that GAPDH is indeed present in the protein-RNA complex resulting from incubation of crude nuclear extracts with a functional region of the PRE. Furthermore, binding of the cellular 35 kDa protein to the PRE fragment is blocked by NAPDH, as would be expected for RNA binding by GAPDH. Finally, purified commercial GAPDH also binds specifically to this RNA fragment. Therefore, GAPDH is one of the cellular proteins that binds to the PRE, and may be involved in the posttranscriptional regulation of hepatitis B virus gene expression.

Expression and analysis of a bacterial poly(hydroxyalkanoate) synthase in insect cells using a baculovirus system.

An improved method for expression of poly-beta-hydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus has been developed using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) in BTI-TN-5B1-4 Trichoplusia ni cells which results in high level production of active PHA synthase. Confirmation of expression of authentic PHA synthase was obtained by Western analysis which also revealed the presence of several apparent proteolytic cleavage products. N-terminal sequence data were obtained from the 64-kDa protein which verified its identity. The PHA synthase produced in this system constitutes approximately 50% of total protein after 60 h of viral infection and is found approximately equally distributed in both soluble and membrane-associated fractions. The expression level allowed rapid purification of the soluble form of PHA synthase to approximately 90% homogeneity in a single liquid chromatography step on hydroxylapatite. Using a direct spectrophotometric assay, analyses show that the enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation between enzyme concentration and specific activity. Over 1000 units of soluble enzyme were obtained from a 250-ml culture of T. ni cells with an apparent initial specific activity of 12 mumol min-1 mg-1. The amount of PHA synthase activity is significantly higher than previously obtained from much larger bacterial cultures. The method described here should provide a general approach for the expression of active PHA synthases from a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing proteins.