On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction.

The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously.

Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era.

The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching.

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry.

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell.

Correlation between protein and mRNA abundance in yeast.

We have determined the relationship between mRNA and protein expression levels for selected genes expressed in the yeast Saccharomyces cerevisiae growing at mid-log phase. The proteins contained in total yeast cell lysate were separated by high-resolution two-dimensional (2D) gel electrophoresis. Over 150 protein spots were excised and identified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Molecular characterization of mitochondrial apoptosis-inducing factor.

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei.

Expression of a tyrosine phosphorylated, DNA binding Stat3beta dimer in bacteria.

The signal transducer and activator of transcription (STAT) proteins deliver signals from the cell membrane to the nucleus. An N-terminally truncated fragment of murine Stat3beta, Stat3betatc (127-722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding.

A simplified gradient solvent delivery system for capillary liquid chromatography-electrospray ionization mass spectrometry.

We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column.

Mapping a protein-binding site on straightened DNA by atomic force microscopy.

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface.

A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multigene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes.

Microfabricated device coupled with an electrospray ionization quadrupole time-of-flight mass spectrometer: protein identifications based on enhanced-resolution mass spectrometry and tandem mass spectrometry data.

We describe the coupling of a microfabricated fluidic device to an electrospray ionization (ESI) quadrupole time-of-flight mass spectrometer (QqTOFMS) for the identification of protein samples. The microfabricated devices consisted of three reservoirs connected via channels to a main capillary, which in turn was linked via a microspray interface to the QqTOFMS. Here we present preliminary results obtained using this system.

Optimization of solid phase microextraction - capillary zone electrophoresis - mass spectrometry for high sensitivity protein identification.

We have previously described the use of a solid phase extraction (SPE) - capillary zone electrophoresis (CZE) - tandem mass spectrometry (MS/MS) system for protein analysis at the low femtomole to subfemtomole level. Here we describe the systematic optimization of a number of parameters which facilitate the use of the SPE-CZE-MS/MS system and further enhance its performance. Specifically, we describe a robust SPE cartridge design which can be assembled without the use of glue, the evaluation of procedures to chemically modify the inner wall of the fused-silica capillaries used in the system to improve separation and reproducibility, and the comparison of different reverse-phase (RP) resins used for the SPE cartridge.

An integrated microfluidics-tandem mass spectrometry system for automated protein analysis.

We describe an integrated analytical system consisting of a microfluidics device micromachined using photolithography/etching technology, a panel of computer-controlled high-voltage relays, and an electrospray ionization tandem mass spectrometer. Movement of solvents and samples on the device and off the device to the mass spectrometer was achieved by directed electroosmotic pumping induced by the activation of a suitable constellation of high-voltage relays. The system was used for the sequential automated analysis of protein digests.

Nanoflow solvent gradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry.

Microfabrication technology offers the opportunity to construct microfluidic modules which are designed to perform specific, dedicated functions. Here we report the construction of a microfabricated device for the generation and delivery by electroosmotic pumping of solvent gradients at nanoliter per minute flow rates. The device consists of three solvent reservoirs and channels which were etched in glass.

RGSZ1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Galphaz phosphorylation, and relationship to a Gz gtpase-activating protein subfamily.

We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J.

Proteome analysis: biological assay or data archive?

In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems.

Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS).

Proteins of rat serum: II. Influence of some biological parameters of the two-dimensional electrophoresis pattern.

This report complements the database already detailed for serum proteins of healthy adult male rats (P. Haynes et al., Electrophoresis 1998, 19, 1484-1492).

Proteins of rat serum: I. Establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.

In the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2-DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography-mass spectrometry (HPLC-MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins.

Characterization of the testis and epididymis in mouse models of human Tay Sachs and Sandhoff diseases and partial determination of accumulated gangliosides.

Beta-hexosaminidase (Hex) is an essential lysosomal enzyme whose activity is higher in the epididymis than in other tissues. The enzyme is also present in sperm and has been postulated to be required for fertilization. To better understand the role of Hex in reproduction, we have examined the testes and epididymides of mouse models of human Tay Sachs and Sandhoff diseases, produced by targeted disruption of the Hexa (alpha-subunit) or Hexb (beta-subunit) genes, respectively, encoding the enzymes Hex A (structure, alphabeta) and Hex B (betabeta).

Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations.

We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80%. A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit.

High sensitivity analysis of proteins and peptides by capillary electrophoresis-tandem mass spectrometry: recent developments in technology and applications.

Analytical biochemistry, in particular the analysis of regulatory proteins that control biological systems and pathways, is dependent on methods of ever-increasing sensitivity. Capillary electrophoresis (CE) has long been recognized as an ultrasensitive analytical technique. In spite of the high sensitivity, CE has not penetrated protein discovery research as a standard analytical method.

Expression and conservation of apolipoprotein AIV in an avian species.

In birds, intestinally derived lipoproteins are thought to be secreted directly into the portal vein rather than to enter the circulation via the lymphatic system as in mammals. Hepatic clearance of these so-called portomicrons must be rapid, but the protein(s) mediating their catabolism, presumably analogues of the 36-kDa mammalian apolipoprotein E, have not been identified. In searching for such a mediator(s), we have isolated a hitherto unknown 38-kDa protein from chicken serum, which we identified by microsequencing and molecular cloning as a counterpart to mammalian apolipoprotein AIV (apoAIV).