The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

The rapid accumulation of sequence data generated by the various genome sequencingprojects and the generation of expressed sequence tag databases has resulted in the need forthe development of fast and sensitive methods for the identification and characterisation oflarge numbers of gel electrophoretically separated proteins to translate the sequence data intobiological function. To achieve this goal it has been necessary to devise new approaches toprotein analysis: matrix-assisted laser desorption and electrospray mass spectrometry havebecome important protein analytical tools which are both fast and sensitive. When combinedwith a robotic system for the in-gel digestion of electrophoretically separated proteins, itbecomes possible to rapidly identify many proteins by searching databases with MS data. Thepower of this combination of techniques is demonstrated by an analysis of the proteins presentin the myofibrillar lattice of the indirect flight muscle of Drosophila melanogaster. Theproteins were separated by SDS-PAGE and in-gel proteolysis was performed bothautomatically and manually. All 16 major proteins could quickly be identified by massspectrometry. Although most of the protein components were known to be present in theflight muscle, two new components were also identified. The combination of methodsdescribed offers a means for the rapid identification of large numbers of gel separatedproteins.     

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.     

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.     

The application of robotics and mass spectrometry to the characterisation of the Drosophila melanogaster indirect flight muscle proteome

Summary The rapid accumulation of sequence data generated by the various genome sequencing projects and the generation of expressed sequence tag databases has resulted in the need for the development of fast and sensitive methods for the identification and characterisation of large numbers of gel electrophoretically separated proteins to translated the sequence data into biological function. To achieve this goal it has been necessary to devise new approaches to protein analysis: matrix-assisted laser desorption and electrospray mass spectrometry have become important protein analytical tools which are both fast and sensitive. When combined with a robotic system for the in-gel digestion of electrophoretically separated proteins, it becomes possible to rapidly identify many proteins by searching databases with MS data. The power of this combination of techniques is demonstrated by an analysis of the proteins present in the myofibrillar lattice of the indirect flight muscle ofDrosophila melanogaster. The proteins were separated by SDS-PAGE and in-gel proteolysis was performed both automatically and manually. All 16 major proteins could quickly be identified by mass spectrometry. Although most of the protein components were known to be present in the flight muscle, two new components were also identified. The combination of methods described offers a means for the rapid identification of large numbers of gel separated proteins.     

Automated Protein Preparation Techniques Using a Digest Robot

Since the introduction of fast analysis methods for peptide mixtures such as MALDI-MS, peptide micropreparation and digest methods have become an important bottleneck in the protein characterization process. We therefore developed and describe here a digest robot capable of processing 30 protein samples in parallel [Houthaeve el al. (1995), FEBS Lett. 376, 91–94]. Briefly, after gel pieces or blots are cut out, they are loaded in flowthrough reactors and these are loaded in a thermocontrolled reactor block. The proteins are then washed, reduced, and alkylated, proteolytically or chemically cleaved, and resulting peptides extracted. The system allows the parallel use of different reagents and enzymes during the same run, and is compatible with RP-HPLC peptide separation and Edman degradation, MALDI-MS, and NanoES-MS/MS. The digest robot is now also commercially available from ABIMED. In an ongoing project aimed at elucidating proteinaceous structures involved in the functional and structural maintenance of the Golgi apparatus, we illustrate the strength of the digest robot for the fast analysis of several proteins. We conclude that the performance of the digest robot is comparable to currently used manual digestion methods. The approach outlined makes sample preparation procedures faster, simpler, and less labor-intensive.