High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry.

We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary. The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion.

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.     

The Anopheles gambiae adult midgut peritrophic matrix proteome

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.     

Consequences of postural changes and removal of vestibular inputs on the movement of air in and out of the lungs of conscious felines.

A variety of experimental approaches in human subjects and animal models established that the vestibular system contributes to regulation of respiration. In cats, the surgical elimination of labyrinthine signals produced changes in the spontaneous activity and posturally related responses of a number of respiratory muscles. However, these effects were complex and sometimes varied between muscle compartments, such that the physiological role of vestibulo-respiratory responses is unclear. The present study determined the functional significance of vestibulo-respiratory influences by examining the consequences of a bilateral labyrinthectomy on breathing rate and the pressure, volume, and flow rate of air exchanged during inspiration and expiration as body orientation with respect to gravity was altered. Data were collected from conscious adult cats acclimated to breathing through a facemask connected to a pneuomotach during 60 degrees head-up pitch and ear-down roll body rotations. Removal of vestibular inputs resulted in a 15% reduction in breathing rate, a 13% decrease in minute ventilation, a 16% decrease in maximal inspiratory airflow rate, and a 14% decrease in the maximal expiratory airflow rate measured when the animals were in the prone position. However, the lesions did not appreciably affect phasic changes in airflow parameters related to alterations in posture. These results suggest that the role of the vestibular system in the control of breathing is to modify baseline respiratory parameters in proportion to the general intensity of ongoing movements, and not to rapidly alter ventilation in accordance with body position.     

Real-time molecular methods to detect infectious viruses

Waterborne transmitted viruses pose a public health threat due to their stability in aquatic environment and the easy transmission with high morbidity rates at low infectious doses. Two major challenge of virus analysis include a lack of adequate information in infectivity and the inability to cultivate certain epidemiologically important viruses in vitro. The use of fluorescent probes in conjunction with fluorescence microscopy allows us to reveal dynamic interactions of the viruses with different cellular structures in living cells that are impossible to detect by immunological or PCR-based experiments. Real-time viral detection in vivo provides sufficient information regarding multiple steps in infection process at molecular level, which will be valuable for the prevention and control of viral infection.     

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore–microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore–microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis.     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.