Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences

Motivation: In any macromolecular polyprotic system - for example protein, DNA or RNA - the isoelectric point - commonly referred to as the pI - can be defined as the point of singularity in a titration curve, corresponding to the solution pH value at which the net overall surface charge - and thus the electrophoretic mobility - of the ampholyte sums to zero. Different modern analytical biochemistry and proteomics methods depend on the isoelectric point as a principal feature for protein and peptide characterization. Protein separation by isoelectric point is a critical part of 2-D gel electrophoresis, a key precursor of proteomics, where discrete spots can be digested in-gel, and proteins subsequently identified by analytical mass spectrometry. Peptide fractionation according to their pI is also widely used in current proteomics sample preparation procedures previous to the LC-MS/MS analysis. Therefore accurate theoretical prediction of pI would expedite such analysis. While such pI calculation is widely used, it remains largely untested, motivating our efforts to benchmark pI prediction methods. Results: Using data from the database PIP-DB and one publically available dataset as our reference gold standard, we have undertaken the benchmarking of pI calculation methods. We find that methods vary in their accuracy and are highly sensitive to the choice of basis set. The machine-learning algorithms, especially the SVM-based algorithm, showed a superior performance when studying peptide mixtures. In general, learning-based pI prediction methods (such as Cofactor, SVM and Branca) require a large training dataset and their resulting performance will strongly depend of the quality of that data. In contrast with Iterative methods, machine-learning algorithms have the advantage of being able to add new features to improve the accuracy of prediction. Contact: [email protected] Availability and Implementation: The software and data are freely available at https://github.com/ypriverol/pIR. Supplementary information: Supplementary data are available at Bioinformatics online

Peptide fractionation for the proteomics studies

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Promiscuous T cell epitopes boosts specific IgM immune response against a P0 peptide antigen from sea lice in different teleost species

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Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with “high-mannose” N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution

A deeper mining on the protein composition of VA-MENGOC-BC®: An OMV-based vaccine against N. meningitidis serogroup B and C

The protein composition of an Outer Membrane Vesicle (OMV) preparation that constitutes the active pharmaceutical ingredient of VA-MENGOC-BC®, an effective vaccine against Neisseria meningitidis serogroups B, and C is presented. This preparation has a high lipid content and five abundant membrane proteins (FetA, PorA, PorB, RmpM, and Opc), constituting approximately 70% of the total protein mass. The protein composition was determined by combining the use of the Hexapeptide Ligand Library and an orthogonal tandem fractionation of tryptic peptides by reverse-phase chromatography at alkaline and acid pH. This approach equalizes the concentration of tryptic peptides derived from low- and high-abundance proteins as well as considerably simplifying the number of peptides analyzed by LC-MS/MS, enhancing the possibility of identifying low-abundance species. Fifty-one percent of the proteins originally annotated as membrane proteins in the genome of the MC58 strain were identified. One hundred and sixty-eight low-abundance cytosolic proteins presumably occluded within OMV were also identified. Four (NadA, NUbp, GNA2091, and fHbp), out of the five antigens constituting the Bexsero® vaccine, were detected in this OMV preparation. In particular, fHbp is also the active principle of the Trumenba® vaccine developed by Pfizer. The HpuA and HpuB gene products (not annotated in the MC58 genome) were identified in the CU385 strain, a clinical isolate that is used to produce this OMV. Considering the proteins identified here and previous work done by our group, the protein catalogue of this OMV preparation was extended to 266 different protein species

Isotopica: a tool for the calculation and viewing of complex isotopic envelopes

The web application Isotopica has been developed as an aid to the interpretation of ions that contain naturally occurring isotopes in a mass spectrum. It allows the calculation of mass values and isotopic distributions based on molecular formulas, peptides/proteins, DNA/RNA, carbohydrate sequences or combinations thereof. In addition, Isotopica takes modifications of the input molecule into consideration using a simple and flexible language as a straightforward extension of the molecular formula syntax. This function is especially useful for biomolecules, which are often subjected to additional modifications other than normal constituents, such as the frequently occurring post-translational modification in proteins. The isotopic distribution of any molecule thus defined can be calculated by considering full widths at half maximum or mass resolution. The combined envelope of several overlapping isotopic distributions of a mixture of molecules can be determined after specifying each molecule's relative abundance. The results can be displayed graphically on a local PC using the Isotopica viewer, a standalone application that is downloadable from the sites below, as a complement to the client browser. The m/z and intensity values can also be obtained in the form of a plain ASCII text file. The software has proved to be useful for peptide mass fingerprinting and validating an observed isotopic ion distribution with reference to the theoretical one, even from a multi-component sample. The web server can be accessed at http://bioinformatica.cigb.edu/isotopica and http://coco.protein.osaka-u.ac.jp/isotopica

Dataset on protein composition of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh 7.5 cells

The four serotypes of dengue virus (DENV1-4) are the causal agents of the emerging disease Dengue Fever and its severe forms. DENV is inoculated into human blood through a mosquito bite. Thus, plasma is an important media for DENV dissemination in infected persons and several important interactions should take place for the virus with human plasma proteins that strongly influence or may determine the course of the infection. This dataset contains 239 proteins identified in the elution fractions of human plasma subjected to DE-52 anion exchange chromatography. Data on DENV2 infection of Huh 7.5 cells in presence of the human plasma fraction is also presented. Keywords: Human plasma, Anion exchange chromatography, Dengue viru

Proteomics and Phospho-Proteomics Profiling of the Co-Formulation of Type I and II Interferons, HeberFERON, in the Glioblastoma-Derived Cell Line U-87 MG

HeberFERON, a co-formulation of Interferon (IFN)-α2b and IFN-γ, has effects on skin cancer and other solid tumors. It has antiproliferative effects over glioblastoma multiform (GBM) clones and cultured cell lines, including U-87 MG. Here, we report the first label-free quantitative proteomic and phospho-proteomic analyses to evaluate changes induced by HeberFERON after 72 h incubation of U-87 MG that can explain the effect on cellular proliferation. LC-MS/MS, functional enrichment and networking analysis were performed. We identified 7627 proteins; 122 and 211 were down- and up-regulated by HeberFERON (fold change > 2; p < 0.05), respectively. We identified 23,549 peptides (5692 proteins) and 8900 phospho-peptides; 523 of these phospho-peptides (359 proteins) were differentially modified. Proteomic enrichment showed IFN signaling and its control, direct and indirect antiviral mechanisms were the main modulated processes. Phospho-proteome enrichment displayed the cell cycle as one of the most commonly targeted events together with cytoskeleton organization; translation/RNA splicing, autophagy and DNA repair, as represented biological processes. There is a high interconnection of phosphoproteins in a molecular network; mTOR occupies a centric hub with interactions with translation machinery, cytoskeleton and autophagy components. Novel phosphosites and others with unknown biological functionality in key players in the aforementioned processes were regulated by HeberFERON and involved CDK and ERK kinases. These findings open new experimental hypotheses regarding HeberFERON action. The results obtained contribute to a better understanding of HeberFERON effector mechanisms in the context of GBM treatment