Proteomes of plasmodium knowlesi early and late ring-stage parasites and infected host erythrocytes.

The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.

A large scale Plasmodium vivax- Saimiri boliviensis trophozoite-schizont transition proteome.

Plasmodium vivax is a complex protozoan parasite with over 6,500 genes and stage-specific differential expression. Much of the unique biology of this pathogen remains unknown, including how it modifies and restructures the host reticulocyte. Using a recently published P. vivax reference genome, we report the proteome from two biological replicates of infected Saimiri boliviensis host reticulocytes undergoing transition from the late trophozoite to early schizont stages. Using five database search engines, we identified a total of 2000 P. vivax and 3487 S. boliviensis proteins, making this the most comprehensive P. vivax proteome to date. PlasmoDB GO-term enrichment analysis of proteins identified at least twice by a search engine highlighted core metabolic processes and molecular functions such as glycolysis, translation and protein folding, cell components such as ribosomes, proteasomes and the Golgi apparatus, and a number of vesicle and trafficking related clusters. Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 enriched functional annotation clusters of S. boliviensis proteins highlighted vesicle and trafficking-related clusters, elements of the cytoskeleton, oxidative processes and response to oxidative stress, macromolecular complexes such as the proteasome and ribosome, metabolism, translation, and cell death. Host and parasite proteins potentially involved in cell adhesion were also identified. Over 25% of the P. vivax proteins have no functional annotation; this group includes 45 VIR members of the large PIR family. A number of host and pathogen proteins contained highly oxidized or nitrated residues, extending prior trophozoite-enriched stage observations from S. boliviensis infections, and supporting the possibility of oxidative stress in relation to the disease. This proteome significantly expands the size and complexity of the known P. vivax and Saimiri host iRBC proteomes, and provides in-depth data that will be valuable for ongoing research on this parasite's biology and pathogenesis.

The Convective Transport of Active Species in the Tropics (CONTRAST) Experiment.

The Convective Transport of Active Species in the Tropics (CONTRAST) experiment was conducted from Guam (13.5° N, 144.8° E) during January-February 2014. Using the NSF/NCAR Gulfstream V research aircraft, the experiment investigated the photochemical environment over the tropical western Pacific (TWP) warm pool, a region of massive deep convection and the major pathway for air to enter the stratosphere during Northern Hemisphere (NH) winter. The new observations provide a wealth of information for quantifying the influence of convection on the vertical distributions of active species. The airborne in situ measurements up to 15 km altitude fill a significant gap by characterizing the abundance and altitude variation of a wide suite of trace gases. These measurements, together with observations of dynamical and microphysical parameters, provide significant new data for constraining and evaluating global chemistry climate models. Measurements include precursor and product gas species of reactive halogen compounds that impact ozone in the upper troposphere/lower stratosphere. High accuracy, in-situ measurements of ozone obtained during CONTRAST quantify ozone concentration profiles in the UT, where previous observations from balloon-borne ozonesondes were often near or below the limit of detection. CONTRAST was one of the three coordinated experiments to observe the TWP during January-February 2014. Together, CONTRAST, ATTREX and CAST, using complementary capabilities of the three aircraft platforms as well as ground-based instrumentation, provide a comprehensive quantification of the regional distribution and vertical structure of natural and pollutant trace gases in the TWP during NH winter, from the oceanic boundary to the lower stratosphere.

Plasmodium vivax trophozoite-stage proteomes.

Plasmodium vivax is the causative infectious agent of 80-300 million annual cases of malaria. Many aspects of this parasite's biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax's known reticulocyte host-cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host-parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function. BIOLOGICAL SIGNIFICANCE: Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host-cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species' known reticulocyte host-cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell's parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.

Biomarkers in pharmacology and drug discovery.

Biomarkers, quantitatively measurable indicators of biological or pathogenic processes, once validated play a critical role in disease diagnostics, the prediction of disease progression, and/or monitoring of the response to treatment. They may also represent drug targets. A number of different methods can be used for biomarker discovery and validation, including proteomics methods, metabolomics, imaging, and genome wide association studies (GWASs) and can be analysed using receiver operating characteristic (ROC) plots. The relative utility of single biomarkers compared to biomarker panels is discussed, along with paradigms for biomarker development, the latter in the context of three large-scale biomarker consortia, the Critical Path Predictive Safety Testing Consortium (PSTC), the NCI Early Detection Research Network (EDRN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). The importance of systematic optimization of many parameters in biomarker analysis, including validation, reproducibility, study design, statistical analysis and avoidance of bias are critical features used by these consortia. Problems including introduction of bias into study designs, data reporting or data analysis are also reviewed.

A systematic quantitative proteomic examination of multidrug resistance in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii strains have been examined at the DNA sequence level, but seldom using large-scale quantitative proteomics. We have compared the proteome of the multidrug resistant strain BAA-1605, with the proteome of the drug-sensitive strain ATCC 17978, using iTRAQ labeling and online 2D LC/MS/MS for peptide/protein identification. Of 1484 proteins present in at least 2 of 4 independent experiments, 114 are 2-fold to 66-fold more abundant in BAA-1605, and 99 are 2-fold to 50-fold less abundant. Proteins with 2-fold or greater abundance in the multidrug resistant strain include drug-, antibiotic-, and heavy metal-resistance proteins, stress-related proteins, porins, membrane transporters, proteins important for acquisition of foreign DNA, biofilm-related proteins, cell-wall and exopolysaccharide-related proteins, lipoproteins, metabolic proteins, and many with no annotated function. The porin CarO, inactivated in carbapenem-resistant strains, is 2.3-fold more abundant in BAA-1605. Likewise, the porin OmpW, less abundant in carbapenem- and colistin-resistant A. baumannii strains, is 3-fold more abundant in BAA-1605. Nine proteins, all present in the drug-sensitive strain but from 2.2-fold to 16-fold more abundant in the MDR strain, can potentially account for the observed resistance of BAA-1605 to 18 antibiotics. BIOLOGICAL SIGNIFICANCE: Multidrug resistant (MDR) strains of the pathogen Acinetobacter baumannii are a significant cause of hospital-acquired infections, are associated with increased mortality and length of stay, and may be a major factor underlying the spread of this pathogen, which is difficult to eradicate from clinical settings. To obtain a better understanding of antimicrobial resistance mechanisms in MDR A. baumannii, we report the first large scale 2D LC/MS/MS-based quantitative proteomics comparison of a drug-sensitive strain and an MDR strain of this pathogen. Ca. 20% of the expressed proteome changes 2-fold or more between the compared strains, including 42 proteins with literature or informatics annotations related to resistance mechanisms, modification of xenobiotics, or drug transport. Other categories of proteins differing 2-fold or more between strains include stress-response related proteins, porins, OMPs, transporters and secretion-related proteins, cell wall- and expolysaccharide-related proteins, lipoproteins, and DNA- and plasmid-related proteins. While the compared strains also differ in other aspects than multi-drug resistance, the observed differences, combined with protein functional annotation, suggest that complex protein expression changes may accompany the MDR phenotype. Expression changes of nine proteins in the MDR strain can potentially account for the observed resistance to 18 antibiotics.

H2B- and H3-specific histone deacetylases are required for DNA methylation in Neurospora crassa.

Neurospora crassa utilizes DNA methylation to inhibit transcription of heterochromatin. DNA methylation is controlled by the histone methyltransferase DIM-5, which trimethylates histone H3 lysine 9, leading to recruitment of the DNA methyltransferase DIM-2. Previous work demonstrated that the histone deacetylase (HDAC) inhibitor trichostatin A caused a reduction in DNA methylation, suggesting involvement of histone deacetylation in DNA methylation. We therefore created mutants of each of the four classical N. crassa HDAC genes and tested their effect on histone acetylation levels and DNA methylation. Global increases in H3 and H4 acetylation levels were observed in both the hda-3 and the hda-4 mutants. Mutation of two of the genes, hda-1 and hda-3, caused partial loss of DNA methylation. The site-specific loss of DNA methylation in hda-1 correlated with loss of H3 lysine 9 trimethylation and increased H3 acetylation. In addition, an increase in H2B acetylation was observed by two-dimensional gel electrophoresis of histones of the hda-1 mutant. We found a similar increase in the Schizosaccharomyces pombe Clr3 mutant, suggesting that this HDAC has a previously unrecognized substrate and raising the possibility that the acetylation state of H2B may play a role in the regulation of DNA methylation and heterochromatin formation.

A cluster-randomized trial to improve stroke care in hospitals.

OBJECTIVE: We evaluated the effect of performance feedback on acute ischemic stroke care quality in Minnesota hospitals. METHODS: A cluster-randomized controlled trial design with hospital as the unit of randomization was used. Care quality was defined as adherence to 10 performance measures grouped into acute, in-hospital, and discharge care. Following preintervention data collection, all hospitals received a report on baseline care quality. Additionally, in experimental hospitals, clinical opinion leaders delivered customized feedback to care providers and study personnel worked with hospital administrators to implement changes targeting identified barriers to stroke care. Multilevel models examined experimental vs control, preintervention and postintervention performance changes and secular trends in performance. RESULTS: Nineteen hospitals were randomized with a total of 1,211 acute ischemic stroke cases preintervention and 1,094 cases postintervention. Secular trends were significant with improvement in both experimental and control hospitals for acute (odds ratio = 2.7, p = 0.007) and in-hospital (odds ratio = 1.5, p < 0.0001) care but not discharge care. There was no significant intervention effect for acute, in-hospital, or discharge care. CONCLUSION: There was no definite intervention effect: both experimental and control hospitals showed significant secular trends with performance improvement. Our results illustrate the potential fallacy of using historical controls for evaluating quality improvement interventions. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that informing hospital leaders of compliance with ischemic stroke quality indicators followed by a structured quality improvement intervention did not significantly improve compliance more than informing hospital leaders of compliance with stroke quality indicators without a quality improvement intervention.

Extensive and varied modifications in histone H2B of wild-type and histone deacetylase 1 mutant Neurospora crassa.

DNA methylation is deficient in a histone deacetylase 1 (HDA1) mutant (hda-1) strain of Neurospora crassa with inactivated histone deacetylase 1. Difference two-dimensional (2D) gels identified the primary histone deacetylase 1 target as histone H2B. Acetylation was identified by LC-MS/MS at five different lysines in wild-type H2B and at 11 lysines in hda-1 H2B, suggesting Neurospora H2B is a complex combination of different acetylated species. Individual 2D gel spots were shifted by single lysine acetylations. FTICR MS-observed methylation ladders identify an ensemble of 20-25 or more modified forms for each 2D gel spot. Twelve different lysines or arginines were methylated in H2B from the wild type or hda-1; only two were in the N-terminal tail. Arginines were modified by monomethylation, dimethylation, or deimination. H2B from wild-type and hda-1 ensembles may thus differ by acetylation at multiple sites, and by additional modifications. Combined with asymmetry-generated diversity in H2B structural states in nucleosome core particles, the extensive modifications identified here can create substantial histone-generated structural diversity in nucleosome core particles.

Neoplasia in the chimpanzee (Pan spp.).

BACKGROUND: Chimpanzees have over 98% genomic sequence homology with humans and may have a similar host response to malignancy. There is minimal information concerning cancer in the chimpanzee and such information would be valuable to individuals caring for and using them for research. METHODS: Spontaneous neoplasia that was documented in two chimpanzee colonies and in the literature were evaluated statistically. RESULTS: In all, 105 spontaneous and 12 experimental neoplasms were diagnosed. Seventy-four spontaneous tumors occurred in females, 24 in males,and seven in animals of undetermined sex. Of the spontaneous tumors 89 were benign, 14 were malignant, and two were undetermined. Neoplasia was most common in the urogenital system in females. CONCLUSIONS: Neoplasia is not uncommon in the chimpanzee, is generally benign, and occurs primarily in the urogenital system in females.

Gas phase dimerization of neuropeptide head activator analogs useful for the noncovalent constraint of peptides.

We have used electrospray mass spectrometry to examine the dimerization of EFLIVKS, a reversed sequence analog of part of the neuropeptide head activator, and other similar analogs. Observation of the EFLIVKS gas phase dimer is concentration-dependent, with a half-saturation concentration for relative dimer formation of 7.8 microM, similar to that of SKVILFE of 12 microM. The lowest energy conformers from quenched molecular dynamics simulations suggest EFLIVKS may dimerize in the gas phase by formation of multiple ion pairs across the dimer interface. Alanine-scan mutants also dimerize in the gas phase, with replacement of the interior residues FLIVK diminishing dimerization. The concentration-dependence of the EFLIVKS circular dichroism spectrum at pH 7.5 suggests the existence of different conformation states at different concentrations, but does not provide evidence supporting the saturable dimer formation in solution. Different analogs of EFLIVKS, when fused to each end of a 18mer unfolded peptide, induce solution structures with T(m)s of 42-50 degrees C. These peptides and analogs may thus be useful for the noncovalent constraint of peptides and peptide library members used in cellular screens.

A soluble 3D LC/MS/MS proteome of the filamentous cyanobacterium Nostoc punctiforme.

Nostoc punctiforme is an oxygenic photoautotrophic cyanobacterium with multiple developmental states, which can form nitrogen-fixing symbioses with a variety of terrestrial plants. 3D LC/MS/MS shotgun peptide sequencing was used to analyze the proteome when N. punctiforme is grown in continuous moderate light with ammonia as the nitrogen source. The soluble proteome includes 1575 proteins, 50% of which can be assigned to core metabolic and transport functions. Another 39% are assigned to proteins with no known function, a substantially higher fraction than in the Escherichia coli proteome. Many expressed proteins protect against oxidative and light stress. Seventy-one sensor histidine kinases, response regulators, and serine/threonine kinases, individually and as hybrid, multidomain proteins, were identified, reflecting a substantial capacity to sense and respond to environmental change. Proteins encoded by each of the five N. punctiforme plasmids were identified, as were 10 transposases, reflecting the plasticity of the N. punctiforme genome. This core proteome sets the stage for comparison with that of other developmental states.

zFP538, a yellow-fluorescent protein from Zoanthus, contains a novel three-ring chromophore.

Crystal structures of the tetrameric yellow-fluorescent protein zFP538 from the button polyp Zoanthus sp. and a green-emitting mutant (K66M) are presented. The atomic models have been refined at 2.7 and 2.5 A resolution, with final crystallographic R factors of 0.206 (R(free) = 0.255) and 0.190 (R(free) = 0.295), respectively, and have excellent stereochemistry. The fold of the protomer is very similar to that of green (GFP) and red (DsRed) fluorescent proteins; however, evidence from crystallography and mass spectrometry suggests that zFP538 contains a three-ring chromophore derived from that of GFP. The yellow-emitting species (lambda(em)(max) = 538 nm) is proposed to result from a transimination reaction in which a transiently appearing DsRed-like acylimine is attacked by the terminal amino group of lysine 66 to form a new six-membered ring, cleaving the polypeptide backbone at the 65-66 position. This extends the chromophore conjugation by an additional double bond compared to GFP, lowering the absorption and emission frequencies. Substitution of lysine 66 with aspartate or glutamate partially converts zFP538 into a red-fluorescent protein, providing additional support for an acylimine intermediate. The diverse and unexpected roles of the side chain at position 66 give new insight into the chemistry of chromophore maturation in the extended family of GFP-like proteins.

Cytomegalovirus-associated discrete gastrointestinal masses in macaques infected with the simian immunodeficiency virus.

Cytomegalovirus (CMV)-associated gastrointestinal masses have been reported in human acquired immune deficiency syndrome patients. This is the first report on CMV-associated gastrointestinal masses in simian immunodeficiency virus (SIV)-infected macaques. Two SIV-infected macaques presented at necropsy with multiple nodular or umbilicated masses within the gastrointestinal tract. In one animal, the masses were located throughout the gastrointestinal tract, whereas in the other, the masses were restricted to the proximal small intestine. Grossly, the masses were indistinguishable from those caused by neoplastic conditions such as lymphoma and, histologically, were composed of hyperplastic glandular tissue, dense neutrophilic infiltrates within the lamina propria, and multifocal proprial hemorrhage. Frequent cytomegalic cells with basophilic intranuclear inclusions were found in affected regions. Immunohistochemistry for CMV demonstrated frequent immunopositive cells within affected areas. Furthermore, immunohistochemistry for the proliferation marker Ki-67 demonstrated increased proliferation in hyperplastic glands and crypts. CMV should be considered a cause of discrete mass lesions in the gastrointestinal tract of SIV-infected macaques.

Riboproteomics of the hepatitis C virus internal ribosomal entry site.

Hepatitis C virus (HCV) protein translation is mediated by a cis-acting RNA, an internal ribosomal entry site (IRES), located in the 5' nontranslated region of the viral RNA. To examine proteins bound to the IRES, which could include proteins important for its function as well as potential drug targets, we used shotgun peptide sequencing to identify proteins in quadruplicate protein affinity extracts of lysed Huh7 cells, obtained using a biotinylated IRES. Twenty-six proteins bound the HCV IRES but not a reversed complementary sequence RNA or vector RNA controls. These included five ribosomal subunits, nine eukaryotic initiation factor 3 subunits, and novel interacting proteins such as the cytoskeletal-related proteins actin, FHOS (formin homologue overexpressed in spleen) and MIP-T3 (microtubule interacting protein that associates with TRAF3). Other novel HCV IRES-binding proteins included UNR (upstream of N-ras), UNR-interacting protein, and the RNA-binding proteins PAI-1 (plasminogen activator inhibitor-1) mRNA binding protein and Ewing sarcoma breakpoint 1 region protein EWS. A large set of additional proteins bound both the HCV IRES and a reversed complementary IRES sequence control, including the known HCV interactors PTB (polypyrimidine tract binding protein), the La autoantigen, and nucleolin. The discovery of these novel HCV IRES-binding proteins suggests links between IRES biology and the cytoskeleton, signal transduction, and other cellular functions.

Multiple functional categories of proteins identified in an in vitro cellular ubiquitin affinity extract using shotgun peptide sequencing.

To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, and the ubiquitin protease inhibitor ubiquitin aldehyde, and was inhibited by iodoacetamide. Quadruplicate affinity extracted proteins were digested with trypsin, and the peptides were analyzed by 2D capillary LC-MS/MS, SEQUEST, MEDUSA, and support vector machine calculations. Identified proteins included 22 proteasome subunits or associated proteins, 18 E1, E2, or E3 ubiquitin system enzymes or related proteins, 4 ubiquitin domain proteins and 36 proteins in functional clusters associated with redox processes, endocytosis/vesicle trafficking, the cytoskeleton, DNA damage/repair, calcium binding, and mRNA splicing. This suggests a link between the ubiquitin system and these cellular processes. This map of cellular ubiquitin-associated proteins may be useful for further studies of ubiquitin system function.

A new algorithm for the evaluation of shotgun peptide sequencing in proteomics: support vector machine classification of peptide MS/MS spectra and SEQUEST scores.

Shotgun tandem mass spectrometry-based peptide sequencing using programs such as SEQUEST allows high-throughput identification of peptides, which in turn allows the identification of corresponding proteins. We have applied a machine learning algorithm, called the support vector machine, to discriminate between correctly and incorrectly identified peptides using SEQUEST output. Each peptide was characterized by SEQUEST-calculated features such as delta Cn and Xcorr, measurements such as precursor ion current and mass, and additional calculated parameters such as the fraction of matched MS/MS peaks. The trained SVM classifier performed significantly better than previous cutoff-based methods at separating positive from negative peptides. Positive and negative peptides were more readily distinguished in training set data acquired on a QTOF, compared to an ion trap mass spectrometer. The use of 13 features, including four new parameters, significantly improved the separation between positive and negative peptides. Use of the support vector machine and these additional parameters resulted in a more accurate interpretation of peptide MS/MS spectra and is an important step toward automated interpretation of peptide tandem mass spectrometry data in proteomics.

Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.

We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.