Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.Current strategy for sequencing proteins in our laboratory by tandem mass spectrometry (1) involves digestion of the protein with site-specific reagents such as cyanogen bromide or trypsin followed by fractionation of the resulting mixture by high-performance liquid chromatography (HPLC). Peptides in each fraction are then ionized by liquid secondary-ion mass spectrometry (LSIMS) (2) on a triple-quadrupole mass spectrometer. Sample is dissolved in a viscous matrix such as glycerol or monothioglycerol and then exposed to a beam of 6-to 8-keV (1 eV = 1.602 x 10-19 J) Cs' ions in the ion source of the mass spectrometer. Peptides are sputtered into the gas phase under these conditions, and the resulting mass spectrum consists largely of (M+H)+ ions characteristic of the molecular weight of each peptide in the sample (3). In a second experiment, the first quadrupole of the instrument is used to select a single (M+H)+ ion from the mixture and to transmit it to quadrupole 2, a collision chamber, where the peptide undergoes collisions with argon atoms and suffers fragmentation primarily at the various amide linkages in the molecule. The resulting fragment ions are then transferred to the third quadrupole, which separates them according to mass. The end result is a mass spectrum containing ions characteristic of the sequence of amino acids in the selected peptide. Repetition of this process under computer control provides sequence information on each peptide in the mixture. Presently, the above approach is limited by the 1800-Da mass range of our triple-quadrupole instrument. Maximum sequence information is obtained only when the protein under investigation is cleaved into peptides of molecular mass under this ceiling. New instrument developments should remedy this situation shortly (4).The major strength of the tandem mass spectrometry method is that it provides extensive sequence information over the whole length of a protein chain in a single series of experiments that involve minimal effort directed toward separation and purification of oligopeptide fragments. Total time for biochemical manipulation, HPLC fractionation, and instrumental analysis of samples from a single protein digest seldom exceeds 4 or 5 days. Even with the limited mass range of our existing instrumentation, it is usually possible ...

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Measles remains an important cause of childhood mortality worldwide. Sustained high vaccination coverage is the key to preventing measles deaths. Because measles vaccine is delivered by injection, hurdles to high coverage include the need for trained medical personnel and a cold chain, waste of vaccine in multidose vials and risks associated with needle use and disposal. Respiratory vaccine delivery could lower these barriers and facilitate sustained high coverage. We developed a novel single unit dose, dry powder live-attenuated measles vaccine (MVDP) for respiratory delivery without reconstitution. We tested the immunogenicity and protective efficacy in rhesus macaques of one dose of MVDP delivered either with a mask or directly intranasal with two dry powder inhalers, PuffHaler and BD Solovent. MVDP induced robust measles virus (MeV)-specific humoral and T-cell responses, without adverse effects, which completely protected the macaques from infection with wild-type MeV more than one year later. Respiratory delivery of MVDP was safe and effective and could aid in measles control.aerosol delivery | protective immunity | multifunctional T cells | antibody avidity M easles is a highly contagious viral disease. Before the availability of measles virus (MeV) vaccines, more than 130 million cases and 7-8 million deaths occurred annually. Intensive immunization efforts with the live attenuated measles vaccine (LAMV) given by injection have resulted in substantial decreases in global measles disease. However, with an estimated 164,000 deaths in 2008 (1), measles continues to be an important cause of child mortality, especially in less-developed regions of the world. The key to prevention of measles is achieving and sustaining high levels of population immunity through vaccination, and substantial challenges for high coverage remain in many countries. Some of the challenges to providing a first dose of measles vaccine to at least 95% of each birth cohort, plus a second dose to older children, are related to the method of vaccine delivery.Measles vaccine is given by injection, and this creates hurdles to sustained high coverage in many developing countries. First, there is often a shortage of the trained personnel needed for sterile reconstitution and safe injection of vaccine. Second, in most developing countries, lyophilized vaccine is in 5-10 dose vials that, after reconstitution, lose 30-50% potency in an hour at 37°C (2), so unused doses must be discarded. Third, contaminated needles and syringes create risks for transmitting bloodborne disease and require safe disposal.As a potential improvement, respiratory delivery of reconstituted liquid LAMV has been studied for more than three decades (3) but has never been licensed or widely used. Aerosol administration of aqueous vaccine is highly effective in boosting preexisting antibody and holds promise for use in older children (4-11), but primary humoral and cellular immune responses vary with the age of vaccinees (12-15).We have developed a dry powder formulation of ...

The aspartate receptor is one of the ligand-specific, homodimeric chemoreceptors that detects extracellular attractants and triggers the chemotaxis pathway of Escherichia coli and Salmonella typhimurium. This receptor regulates the activity of the histidine kinase CheA, which forms a kinetically stable complex with the receptor cytoplasmic domain. An atomic four-helix bundle model has been constructed for this domain, which is functionally subdivided into the signaling and adaptation subdomains. The proposed four-helix bundle structure of the signaling subdomain, which binds CheA, is fully supported by experimental evidence. Much less evidence is available to test the four-helix bundle model of the adaptation subdomain, which possesses covalent adaptation sites and docking surfaces for adaptation enzymes. The present study focuses on a putative helix near the C terminus of the adaptation subdomain. To probe the structural and functional features of positions G467-A494 in this C-terminal region, a cysteine and disulfide scanning approach has been employed. Measurement of the chemical reactivities of scanned cysteines reveals an α-helical periodicity of exposed and buried residues, confirming α-helical secondary structure and mapping out a buried packing face. The effects of cysteine substitutions on activity in vivo and in vitro highlight the functional importance of the helix, especially its buried face. A scan for disulfide bond formation between symmetric pairs of engineered cysteines reveals promiscuous collisions between subunits, indicating the presence of significant thermal dynamics. A scan for functional disulfides reveals lockon and signal-retaining disulfide bonds formed between symmetric pairs of cysteines at buried positions, indicating that the buried face of the helix lies near the subunit interface of the homodimer in the equilibrium structures of both the apo and aspartate-bound states where it plays a critical role in kinase regulation. These results strongly support the existing four-helix bundle model of the adaptation subdomain structure. A mechanistic model is proposed in which a signal is transmitted through the adaptation subdomain by a change in supercoiling of the four-helix bundle.The mechanism of signal transduction by which cell-surface receptors regulate cytoplasmic kinases is an issue of central importance in signaling biology. Some receptors activate their appropriate kinases by dimerization, but others trigger kinase activation by transmembrane conformational changes. The latter class includes a large superfamily of cell-surface receptors that regulate cytoplasmic histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (1,2). An important subfamily of this histidine kinase-coupled receptor superfamily is responsible for regulating the thermo-, photo-, osmo-, redox-, and chemotaxis pathways of a wide variety of prokaryotic organisms (3-5). These taxis receptors, comprising a group of over 2000 homologues (6), exhibit regions of high conservation in ...