Abetting host immune response by inhibiting rhipicephalus sanguineus Evasin-1: An in silico approach.

The brown dog tick (Rhipicephalus sanguineus) is the most prevalent tick in the world and a well-recognized vector of many pathogens affecting dogs and occasionally humans. Pathogens exploit tick salivary molecules for their survival and multiplication in the vector and transmission to and establishment in the hosts. Tick saliva contains various non-proteinaceous substances and secreted proteins that are differentially produced during feeding and comprise of inhibitors of blood congealing and platelet aggregation, vasodilatory and immunomodulatory substances, and compounds preventing itch and pain. One of these proteins is Evasin-1, which has a high binding affinity to certain types of chemokines. The binding of Evasin-1 to chemokines prevents the detection and immune response of the host to R. sanguineus, which may result in the successful transmission of pathogens. In this study, we screened potential Evasin-1 inhibitor based on the pharmacophore model derived from the binding site residues. Hit ligands were further screened via molecular docking and virtual ADMET prediction, which resulted in ZINC8856727 as the top ligand (binding affinity: -9.1 kcal/mol). Molecular dynamics simulation studies, coupled with MM-GBSA calculations and principal component analysis revealed that ZINC8856727 plays a vital role in the stability of Evasin-1. We recommend continuing the study by developing a formulation that serves as a potential medicine aid immune response during R. sanguineus infestation.

Critically Ill COVID-19 Patients Exhibit Anti-SARS-CoV-2 Serological Responses.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a global health care emergency. Anti-SARS-CoV-2 serological profiling of critically ill COVID-19 patients was performed to determine their humoral response. Blood was collected from critically ill ICU patients, either COVID-19 positive (+) or COVID-19 negative (-), to measure anti-SARS-CoV-2 immunoglobulins: IgM; IgA; IgG; and Total Ig (combined IgM/IgA/IgG). Cohorts were similar, with the exception that COVID-19+ patients had a greater body mass indexes, developed bilateral pneumonias more frequently and suffered increased hypoxia when compared to COVID-19- patients (p < 0.05). The mortality rate for COVID-19+ patients was 50%. COVID-19 status could be determined by anti-SARS-CoV-2 serological responses with excellent classification accuracies on ICU day 1 (89%); ICU day 3 (96%); and ICU days 7 and 10 (100%). The importance of each Ig isotype for determining COVID-19 status on combined ICU days 1 and 3 was: Total Ig, 43%; IgM, 27%; IgA, 24% and IgG, 6%. Peak serological responses for each Ig isotype occurred on different ICU days (IgM day 13 > IgA day 17 > IgG persistently increased), with the Total Ig peaking at approximately ICU day 18. Those COVID-19+ patients who died had earlier or similar peaks in IgA and Total Ig in their ICU stay when compared to patients who survived (p < 0.005). Critically ill COVID-19 patients exhibit anti-SARS-CoV-2 serological responses, including those COVID-19 patients who ultimately died, suggesting that blunted serological responses did not contribute to mortality. Serological profiling of critically ill COVID-19 patients may aid disease surveillance, patient cohorting and help guide antibody therapies such as convalescent plasma.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

An inducible mouse model of podocin-mutation-related nephrotic syndrome.

Mutations in the NPHS2 gene, encoding podocin, cause hereditary nephrotic syndrome. The most common podocin mutation, R138Q, is associated with early disease onset and rapid progression to end-stage renal disease. Knock-in mice carrying a R140Q mutation, the mouse analogue of human R138Q, show developmental arrest of podocytes and lethal renal failure at neonatal age. Here we created a conditional podocin knock-in model named NPHS2 R140Q/-, using a tamoxifen-inducible Cre recombinase, which permits to study the effects of the mutation in postnatal life. Within the first week of R140Q hemizygosity induction the animals developed proteinuria, which peaked after 4-5 weeks. Subsequently the animals developed progressive renal failure, with a median survival time of 12 (95% CI: 11-13) weeks. Foot process fusion was observed within one week, progressing to severe and global effacement in the course of the disease. The number of podocytes per glomerulus gradually diminished to 18% compared to healthy controls 12-16 weeks after induction. The fraction of segmentally sclerosed glomeruli was 25%, 85% and 97% at 2, 4 and 8 weeks, respectively. Severe tubulointerstitial fibrosis was present at later disease stage and was correlated quantitatively with the level of proteinuria at early disease stages. While R140Q podocin mRNA expression was elevated, protein abundance was reduced by more than 50% within one week following induction. Whereas miRNA21 expression persistently increased during the first 4 weeks, miRNA-193a expression peaked 2 weeks after induction. In conclusion, the inducible R140Q-podocin mouse model is an auspicious model of the most common genetic cause of human nephrotic syndrome, with a spontaneous disease course strongly reminiscent of the human disorder. This model constitutes a valuable tool to test the efficacy of novel pharmacological interventions aimed to improve podocyte function and viability and attenuate proteinuria, glomerulosclerosis and progressive renal failure.

De Novo Sequencing Assisted Approach for Characterizing Mixture MS/MS Spectra.

Extensive research has been conducted for the computational analysis of mass spectrometry based proteomics data. However, there are still remaining challenges, among which, one particular challenge is the low identification rate of the collected spectral data. A specific contributing factor is the existence of mixture spectra in the collected MS/MS spectra which are generated by the concurrent fragmentation of multiple precursors in one sequencing attempt. The quite frequently observed mixture spectra necessitates the development of effective computational approaches to characterize those non-conventional spectral data. In this research, we proposed an approach for matching the query mixture spectra with a pair of peptide sequences acquired from the protein database by incorporating a special de novo assisted filtration strategy. The experiment results on two different datasets of MS/MS spectra containing mixed ion fragments from multiple peptides demonstrated the efficiency of the integrated filtration strategy in reducing examination space and verified the effectiveness of the proposed matching scheme as well.

Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania.

Campylobacter species are commonly transmitted to humans through consumption of contaminated foods such as milk and meat. The aim of this study was to investigate the prevalence, antimicrobial resistance, and genetic determinants of resistance of Campylobacter isolated from raw milk and beef carcasses in Tanzania. The antimicrobial resistance genes tested included blaOXA-61 (ampicillin), aph-3-1 (aminoglycoside), tet(O) (tetracycline), and cmeB (multi-drug efflux pump). The prevalence of Campylobacter was 9.5% in beef carcasses and 13.4% in raw milk, respectively. Using multiplex-polymerase chain reaction (PCR), we identified 58.1% of the isolates as Campylobacter jejuni, 30.7% as Campylobacter coli, and 9.7% as other Campylobacter spp. One isolate (1.6%) was positive for both C. jejuni and C. coli specific PCR. Antimicrobial susceptibility testing using the disk diffusion assay and the broth microdilution method showed resistance to: ampicillin (63% and 94.1%), ciprofloxacin (9.3% and 11.8%), erythromycin (53.7% and 70.6%), gentamicin (0% and 15.7%), streptomycin (35.2% and 84.3%), and tetracycline (18.5% and 17.7%), respectively. Resistance to azithromycin (42.6%), nalidixic acid (64.8%), and chloramphenicol (13%) was determined using the disk diffusion assay only, while resistance to tylosin (90.2%) was quantified using the broth microdilution method. The blaOXA-61 (52.6% and 28.1%), cmeB (26.3% and 31.3%), tet(O) (26.3% and 31.3%), and aph-3-1 (5.3% and 3.0%) were detected in C. coli and C. jejuni. These findings highlight the extent of antimicrobial resistance in Campylobacter occurring in important foods in Tanzania. The potential risks to consumers emphasize the need for adequate control approaches, including the prudent use of antimicrobials to minimize the spread of antimicrobial-resistant Campylobacter.

Spatial predictors of bovine tuberculosis infection and Brucella spp. exposure in pastoralist and agropastoralist livestock herds in the Ruaha ecosystem of Tanzania.

While many studies investigate animal-related risk factors for disease, few have considered environmental or spatial risk factors in the dynamics of bovine tuberculosis (bTB) and brucellosis. In the Ruaha ecosystem of Tanzania, we investigated the role of household location as a predictor for infection with Mycobacterium bovis and exposure to Brucella in pastoralist and agropastoralist cattle herds in a typical African wildlife-livestock-human interface. ArcGIS was utilized to calculate Euclidian distances between households and the nearest river, village center, protected area, and other infected households, followed by multivariate logistic regression to assess the association between risk factors and herd-level bTB and Brucella outcomes. Global and local spatial clustering of bTB-infected and Brucella-exposed herds was explored using the Cuzick-Edward's test and SaTScan spatial scan statistics. Households located farther from rivers and closer to village centers and herds belonging to agropastoralists were more likely to have bTB-positive cattle. Risk of Brucella exposure increased with proximity to protected areas. One spatial cluster of households with Brucella spp. seropositive cattle was identified. Spatial factors may be useful for assessing disease risk and for formulating intervention and control strategies for households that manage cattle in ecosystems characterized by seasonally limited resources and intense wildlife-livestock interfaces.

Profile of plasma lipids and degree of derangements among the elderly of Morogoro region, Tanzania.

Changes in lifestyles and ageing have been associated with growing rates of modifiable cardiovascular risk factors (CRF). Dyslipidemia is one ofthe CRF associated with numbers of cardiovascular diseases. This descriptive cross-sectional study was conducted to determine the profile and degree of derangements of plasma lipids among 300 (176 females and 124 males) elderly individuals aged ≥ 60 years in Morogoro, Tanzania. The calorimetric enzymatic methods and the Friedewal's equation were used for determination of cholesterols and triglycerides (TG). Social and demographic characteristics were gathered by structured questionnaires. The logistic regression models were used to identify the determinants of abnormal serum lipids level. Mean Total Cholesterols (TC) and Low Density Lipoprotein Cholesterols (LDL-C) in females exceeded significantly that of males. Mean TC, LDL-C as well as TG (mg/dL) declined significantly with age while mean High Density Lipoprotein Cholesterols (HDL-C) also declined but only slightly. Elderly females were two times more likely to have elevated TC (OR = 2.11; 95% CI: 1.04-4.28: P = 0.05) and LDL-C (OR = 2.15; 95% CI: 1.17- 3.97: P = 0.019) and three times to have lowered HDL-C (OR = 3; 95% CI: 1.97-5.30: P < 0.001) than males. Urban residents were about two times more likely to have elevated LDL-C (OR = 1.84; 95% CI: 1.04-3.25: P = 0.047) than their rural counterparts. Body Mass Index of ≥ 30 kg/m2 was also associated with elevated LDL-C (OR = 1.89; 95% CI: 1.05-3.42: P = 0.045) and lowered HDL-C (OR = 2.18; 95% CI: 1.3-3.65: P = 0.004), respectively. The present study has established the profile and level of derangements of serum lipids among the elderly of Morogoro region in Tanzania. It appears that, female sex and BMI of ≥ 30 kg/m2 are significant factors for elevated TC, LDL-C and lowered HDL-C while urban life is a significant factor for elevated LDL-C.

Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry.

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Silk cocoon of Bombyx mori: proteins and posttranslational modifications--heavy phosphorylation and evidence for lysine-mediated cross links.

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2-DE was carried out with multi-enzyme in-gel digestion followed by MS identification of silk-peptides. High-sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys->allysine formation have been observed providing evidence for lysine-mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di-tyrosine cross links. A high degree of sequence conflicts probably representing single-nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.

Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells.

Manganese superoxide dismutase (Mn-SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn-SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2-DE, multienzyme digestions were carried out and peptides were analyzed by MALDI-TOF/TOF, Qq-TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp-186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2-oxo-histidine and a previously described intermediate (His+14). Complete sequencing of the two Mn-SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn-SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn-SOD activity.

Hippocampal synapsin isoform levels are linked to spatial memory enhancement by SGS742.

Synapsins are essential proteins for synaptic plasticity and there is no information available for their role in cognitive enhancement (CE) of spatial memory formation. It was therefore the aim of the study to link individual synapsin proteins and their isoforms to spatial memory formation enhanced by SGS742 in the mouse. Extracted hippocampal proteins from a cognitive study treating OF1 mice with the cognitive enhancer SGS742 and tested in the Morris water maze, were run on two-dimensional gel electrophoresis. Subsequently, protein spots were unambiguously identified by qQ-TOF mass spectrometry. Quantification of proteins from four groups (NaCl-treated mice, SGS742-treated mice, SGS742-treated yoked controls, and NaCl-treated yoked controls) was carried out according to an in-gel stable isotope labeling method. A total of 17 protein spots representing synapsin isoforms were identified and quantified. Using quantification of individual synapsin isoforms showed that these can be clearly assigned to CE by the GABAB antagonist SGS742. Quantitative determination of individual synapsin isoform showed an increase in SGS742-treated mice (mean+/-SD) of ratios between light and heavy stable isotope labeled synapsin protein (SGS742 vs. controls: 2.19+/-0.41 for synapsin Ia, and 1.41+/-0.81 for synapsin IIa). Synapsins Ib and IIb were not linked to CE. The NaCl-treated controls and the use of yoked controls that were ruling out swimming- and stress-mediated changes of synapsins, unequivocally allow to propose a role for synapsins Ia and IIa in the mechanism of CE of spatial memory formation.

Neurotrophin 3/TrkC-regulated proteins in the human medulloblastoma cell line DAOY.

Medulloblastoma (MB) is the most common malignant childhood brain tumor and high neurotrophin (NP) receptor TrkC mRNA expression was identified as a powerful independent predictor of favorable survival outcome. In order to determine downstream effector proteins of TrkC signaling, the MB cell line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. A proteomic approach was used to search for expressional changes by two mass spectrometric methods and immunoblotting for validation of significant results. Multiple time points for up to 48 h following NP-3-induced TrkC receptor activation were chosen. Thirteen proteins from several pathways (nucleoside diphosphate kinase A, stathmin, valosin-containing protein, annexin A1, dihydropyrimidinase-related protein-3, DJ-1 protein, glutathione S-transferase P, lamin A/C, fascin, cofilin, vimentin, vinculin, and moesin) were differentially expressed and most have been shown to play a role in differentiation, migration, invasion, proliferation, apoptosis, drug resistance, or oncogenesis. Knowledge on effectors of TrkC signaling may represent a first useful step for the identification of marker candidates or reflecting probable pharmacological targets for specific treatment of MB.

Mass spectrometric studies on mouse hippocampal synapsins Ia, IIa, and IIb and identification of a novel phosphorylation site at serine-546.

Synapsins are key phosphoproteins in the mammalian brain, and structural research on synapsins is still holding center stage. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis (2-DE), and the spots were analyzed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS. Synapsins Ia, IIa, and IIb were unambiguously identified and represented by 15 individual spots on 2-DE. Several serine phosphorylation sites were confirmed, and a novel phosphorylation site was observed at Ser-546 in synapsin IIa in all gels analyzed.

Nitric oxide and oxygen radical attack on GDP-dissociation inhibitor 2 (GDI-2) in spinal cord injury of the rat.

Protein oxidation and nitration have been described during spinal cord injury (SCI) in animal models. Herein, mass spectrometry unambiguously identified GDP-dissociation inhibitor-2 (GDI-2) in SCI with post-translational modifications of 3-aminotyrosine (8 h post-injury) and an acrolein adduct of GDI-2 (72 h post-injury). On the basis of mass spectrometry evidence, we conclude that lipid-peroxidation and protein nitration do take place on an important signalling protein that may be prevented by specific experimental therapeutic interventions.

Proteomic analysis of rat cerebral cortex, hippocampus and striatum after exposure to morphine.

Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.

An integrated map of the murine hippocampal proteome based upon five mouse strains.

With the advent of proteomics technologies it is possible to simultaneously demonstrate the expression of hundreds of proteins. The information offered by proteomics provides context-based understanding of cellular protein networks and has been proven to be a valuable approach in neuroscience studies. The mouse hippocampus has been a major target of analysis in the search for molecular correlates to neuronal information storage. Although human and rat hippocampal samples have been successfully subjected to proteomic profiling, no elaborate analysis providing the fundamental experimental basis for protein-expression studies in the mouse hippocampus has been carried out as yet. This led us to construct a master map generated from the individual hippocampal proteomes of five different mouse strains. A proteomic approach, based upon 2-DE coupled to MS (MALDI-TOF/TOF) has been chosen in an attempt to establish a comprehensive reference database of proteins expressed in the mouse hippocampus. 469 individual proteins, represented by 1156 spots displaying various functional states of the respective gene products were identified. Proteomic profiling of the hippocampus, a brain region with a pivotal role for neuronal information processing and storage may provide insight into the characteristics of proteins serving this highly sophisticated function.

Components of the protein quality control system are expressed in a strain-dependent manner in the mouse hippocampus.

Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains.

Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure.

Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance.

Searching for hypothetical proteins: theory and practice based upon original data and literature.

A large part of mammalian proteomes is represented by hypothetical proteins (HP), i.e. proteins predicted from nucleic acid sequences only and protein sequences with unknown function. Databases are far from being complete and errors are expected. The legion of HP is awaiting experiments to show their existence at the protein level and subsequent bioinformatic handling in order to assign proteins a tentative function is mandatory. Two-dimensional gel-electrophoresis with subsequent mass spectrometrical identification of protein spots is an appropriate tool to search for HP in the high-throughput mode. Spots are identified by MS or by MS/MS measurements (MALDI-TOF, MALDI-TOF-TOF) and subsequent software as e.g. Mascot or ProFound. In many cases proteins can thus be unambiguously identified and characterised; if this is not the case, de novo sequencing or Q-TOF analysis is warranted. If the protein is not identified, the sequence is being sent to databases for BLAST searches to determine identities/similarities or homologies to known proteins. If no significant identity to known structures is observed, the protein sequence is examined for the presence of functional domains (databases PROSITE, PRINTS, InterPro, ProDom, Pfam and SMART), subjected to searches for motifs (ELM) and finally protein-protein interaction databases (InterWeaver, STRING) are consulted or predictions from conformations are performed. We here provide information about hypothetical proteins in terms of protein chemical analysis, independent of antibody availability and specificity and bioinformatic handling to contribute to the extension/completion of protein databases and include original work on HP in the brain to illustrate the processes of HP identification and functional assignment.