The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in < 5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic-aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic-aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.

Neuropeptides are important messenger molecules that influence nearly all physiological processes. In insects, they can be released as neuromodulators within the central nervous system (CNS) or as neurohormones into the hemolymph. We analyzed the peptidome of neurohormonal release sites and associated secretory peptidergic neurons of adult Drosophila melanogaster. MALDI-TOF mass spectrometric analyzes were performed on single organs or cell cluster from individual flies. This first peptidomic characterization in adult fruit flies revealed 32 different neuropeptides. Peptides not directly predictable from previously cloned or annotated precursor genes were sequenced by tandem mass spectrometry. These peptides turned out to be either intermediate products of neuropeptide processing or shorter versions of known peptides. We found that the peptidome of the CNS-associated neurohemal organs is tagma-specific in Drosophila. Abdominal neurohemal organs and their supplying peptidergic neurons contain the capa gene products periviscerokinins and pyrokinin-1, thoracic neurohemal organs contain FMRFamides, and the neurohemal release sites of the brain contain pyrokinin-1(2-15), pyrokinin-2, corazonin, myosuppressin, and sNPF as their major putative release products. Our results show that peptidomic approaches are well suited to study differential neuropeptide expression or posttranslational modifications in morphologically defined parts of the nervous system and in a developmental and physiological context in animals as small as Drosophila melanogaster.

The occlusion derived form of baculovirus is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. Because knowledge of virion composition is a prerequisite for functional investigation, this study used a combination of techniques to identify the proteins present within or associated with the occlusion-derived virus (ODV) virion. Thirtyone proteins, including proteins known to be essential for viral DNA replication, were identified with confidence. An additional 13 proteins were identified by using one of the three techniques. A comparison of gene conservation among the ODV proteins encoded in the 16 sequenced baculoviridae genomes is presented. With knowledge of the composition of ODV, it is now possible to target proteins and study their role(s) during primary infection.A utographa californica nucleopolyhedrovirus (AcMNPV) is the type species for the family Baculoviridae and it was used in this study. AcMNPV is a double-stranded DNA virus (132 kbp) that undergoes a biphasic life cycle in its lepidopteron host. Progeny nucleocapsids have two fates: during the early phase of infection, Ϸ16% of the intracellular copies of viral DNA are targeted for maturation at the cell surface to produce budded virus (BV) (1). The remaining nucleocapsids mature within the nucleus and are incorporated within a viral occlusion (occlusionderived virus, ODV). After primary infection of the insect gut by ODV, BV is produced and released into the hemocoel, and secondary infection results in insect death with subsequent release of viral occlusions into the environment. Because ODV is the viral form responsible for primary infection, knowing virion composition is fundamental for functional investigation of virulence and host specificity. The goal of this study was to determine the protein composition of the ODV virion. Such knowledge should aid in the understanding of the biology of AcMNPV, including genetic manipulation of the family Baculoviridae to enhance their function as microbial pesticides (2). Additionally, studies on the mechanism of envelope protein trafficking to intranuclear membranes and ODV envelope would be aided by comprehensive knowledge of ODV envelope composition.ODV is amenable to proteomic approaches for protein identification. It is easily purified and contains a small number of proteins. Previous studies suggest ODV contains between 13 and 35 proteins, most of which are unknown (3-13). ODV is incorporated within a crystalline occlusion, and the increased density of the occlusion allows it to be easily purified from in vitro or in vivo sources. A major concern when releasing the ODV from the crystalline matrix is protein degradation caused by the presence of proteases, particularly an insect alkaline protease (14). To inhibit protease activity, the occlusions are treated with HgCl 2 or diisopropyl fluorophosphate (14). After protease inactivation, ODV is released from the occlusion an...

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component ␣-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell. Molecular & Cellular Proteomics 4:56 -72, 2005.

Two's company: Using a wild‐type or evolved PylRS‐pylTUUA pair to suppress ochre mutation and an evolved MjTyrRS‐Mj${{{\rm {\rm tRNA}}{{{\rm {\rm Tyr}}\hfill \atop {\rm {\rm CUA}}\hfill}}}}$ pair to suppress amber mutation, two different noncanonical amino acids (NAAs) have been concomitantly incorporated into one protein in E. coli with high efficiency (see picture, with NAAs 1–4; GFP=green‐fluorescent protein).