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Context Glucose-dependent insulinotropic polypeptide (GIP) has been proposed to exert insulin-independent effects on lipid and bone metabolism. Objective We investigated the effects of a 6-day subcutaneous GIP infusion on circulating lipids, white adipose tissue (WAT), brown adipose tissue (BAT), hepatic fat content, inflammatory markers, respiratory exchange ratio (RER), and bone homeostasis in patients with type 1 diabetes. Methods In a randomized, placebo-controlled, double-blind, crossover study, 20 men with type 1 diabetes underwent a 6-day continuous subcutaneous infusion with GIP (6 pmol/kg/min) and placebo (saline), with an interposed 7-day washout period. Results During GIP infusion, participants (26 ± 8 years [mean ± SD]; BMI 23.8 ± 1.8 kg/m2; glycated hemoglobin A1c 51 ± 10 mmol/mol [6.8 ± 3.1%]) experienced transiently increased circulating concentrations of nonesterified fatty acid (NEFA) (P = 0.0005), decreased RER (P = 0.009), indication of increased fatty acid β-oxidation, and decreased levels of the bone resorption marker C-terminal telopeptide (P = 0.000072) compared with placebo. After 6 days of GIP infusion, hepatic fat content was increased by 12.6% (P = 0.007) and supraclavicular skin temperature, a surrogate indicator of BAT activity, was increased by 0.29 °C (P < 0.000001) compared with placebo infusion. WAT transcriptomic profile as well as circulating lipid species, proteome, markers of inflammation, and bone homeostasis were unaffected. Conclusion Six days of subcutaneous GIP infusion in men with type 1 diabetes transiently decreased bone resorption and increased NEFA and β-oxidation. Further, hepatic fat content, and supraclavicular skin temperature were increased without affecting WAT transcriptomics, the circulating proteome, lipids, or inflammatory markers.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10 -7 mol·L -1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Breast cancer is the most prevalent cancer in women worldwide. Its molecular subtypes are based on the presence/absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). MACL-1 and MGSO-3 are cell lines derived from primary tumor sites of patients diagnosed with luminal A subtype carcinoma (ER+/PR+/HER2-) and ductal carcinoma in situ (ER-/PR-/HER2+), respectively. However, these cell lines lost the expression of these markers over cell culturing, and both have triple-negative phenotypes (ER-/PR-/HER2-), which has the poorest prognosis. Here, we sought to study the proteome signature of MGSO-3 and MACL-1, comparing them with the epithelial cell line MCF-10A and the well-established metastatic-derived breast cancer cell line MDA-MB-231. Our results showed that proteins associated with the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) were upregulated in MGSO-3 and MACL-1 cells. These cell lines also showed upregulation of pro-apoptotic proteins when compared with MDA-MB-231. The molecular differences highlighted in this study may clarify the molecular basis behind cancer cells functioning and may reveal novel signatures across the breast cancer cell models.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

On-tissue digestion has become the preferred method to identify proteins in mass spectrometry (MS) imaging. In this study, we report advances in data acquisition and protein identification for MS imaging after on-tissue digestion. Tryptic peptides in a coronal mouse brain section were measured at 50 μm pixel size and revealed detailed histological structures, e.g., the ependyma (consisting of one to two cell layers), which was confirmed by H&E staining. This demonstrates that MS imaging of tryptic peptides at or close to cellular resolution is within reach. We also describe a detailed identification workflow which resulted in the identification of 99 proteins (with 435 corresponding peptides), based on comparison with LC-MS/MS data and in silico digest. These results were obtained with stringent parameters, including high mass accuracy in imaging mode (RSME

The 2020 European Bioinformatics Community for Mass Spectrometry (EuBIC-MS) Developers’ meeting was held from January 13th to January 17th 2020 in Nyborg, Denmark. Among the participants were scientists as well as developers working in the field of computational mass spectrometry (MS) and proteomics. The 4-day program was split between introductory keynote lectures and parallel hackathon sessions. During the latter, the participants developed bioinformatics tools and resources addressing outstanding needs in the community. The hackathons allowed less experienced participants to learn from more advanced computational MS experts, and to actively contribute to highly relevant research projects. We successfully produced several new tools that will be useful to the proteomics community by improving data analysis as well as facilitating future research. All keynote recordings are available on https://doi.org/10.5281/zenodo.3890181.

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications.

Streptomycetes are multicellular bacteria with complex developmental cycles. They are of biotechnological importance as they produce most bioactive compounds used in biomedicine, e.g. antibiotic, antitumoral and immunosupressor compounds. Streptomyces genomes encode many Ser/Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (first mycelium); secondary metabolite producing hyphae (second mycelium); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signaling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism. Phosphoproteomics revealed 85 unique protein phosphorylation sites, 58 of them differentially phosphorylated during differentiation. Computational analysis suggested that these regulated protein phosphorylation events are implicated in important cellular processes, including cell division, differentiation, regulation of secondary metabolism, transcription, protein synthesis, protein folding and stress responses. We discovered a novel regulated phosphorylation site in the key bacterial cell division protein FtsZ (pSer319) that modulates sporulation and regulates actinorhodin antibiotic production. We conclude that manipulation of distinct protein phosphorylation events may improve secondary metabolite production in industrial streptomycetes, including the activation of cryptic pathways during the screening for new secondary metabolites from streptomycetes.

The inaugural European Bioinformatics Community (EuBIC) developer's meeting was held from January 9th to January 12th 2018 in Ghent, Belgium. While the meeting kicked off with an interactive keynote session featuring four internationally renowned experts in the field of computational proteomics, its primary focus were the hands-on hackathon sessions which featured six community-proposed projects revolving around three major topics: (i) quality control; (ii) workflows, protocols, and guidelines; and (iii) quantification. Here, we present an overview of the scientific program of the EuBIC developer's meeting and provide a starting point for follow-up on the covered projects.

Lignosulfonates are abundantly available byproducts of the paper and pulping industry, and they therefore represent a promising feedstock for new sustainable processes. For industrial applications of lignosulfonates, their molecular weight distribution is a critical factor. In order to decrease the average molecular weight of lignosulfonates, Seventeen basidiomycetes were screened for their capability to depolymerize lignosulfonates from spent sulfite liquor (SSL) in surface and liquid cultures. Five basidiomycetes polymerized the lignosulfonates under the selected conditions. Only Irpex consors was found to efficiently degrade calcium lignosulfonates when SSL (0.5%, w/w) was used as the sole carbon and nitrogen source. The average molecular weight of the lignosulfonates was reduced from ∼26 to ∼4 kDa as determined by size exclusion chromatography (SEC) within two weeks. Various extracellular enzyme activities of I. consors were determined over the culture period. High peroxidase activities were correlating with a high degradation rate and the culture was harvested at the day of highest peroxidase activity. A putative versatile peroxidase was isolated by fast protein liquid chromatography (FPLC) and its encoding cDNA was cloned.

Insects are able to feed on a broad spectrum of nutritional sources, due to a variable enzymatic system which can be endogenic or provided by associated microorganisms. This enzymatic system may be employed for the hydrolysis of industrial relevant proteins. Several grain pests were screened for their ability to hydrolyze storage proteins from wheat and rice as well as casein. Zymograms identified hydrolytic activities of the lesser grain borer Rhizopertha dominica against gluten and rice protein. Besides, R. dominica showed the highest prolyl-specific peptidase activity among all tested insects. Enzyme extracts of R. dominica were purified via anion exchange chromatography using a fast protein liquid chromatography system. Two of the purified peptidase fractions were able to hydrolyze peptides from wheat and barley relevant for celiac disease showing a proline preferential cleaving pattern.

A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics.

Skin secretions of amphibian are an interesting source of biologically active peptides. The present study provides the profile of the skin secretions of the brown frog Rana temporaria from Zvenigorod population (Russia). Sequencing of the skin secretion components has been carried out on an ion cyclotron resonance instrument with electrospray ionization and two methods of fragmentation activation, collisional activation and electron capture. For sequencing of the peptides containing intermolecular C-terminal disulfide cycle two methods of disulfide bond opening have been used: reduction with subsequent alkylation of the free thiol groups and oxidation with performic acid with the formation of sulfo-acid groups. The peptide profile of Rana temporaria studied by a complex mass spectral method has been compared with the data for the frogs of other European populations of this species. For the first time we have revealed ornithokinin-antagonist of the ornithokinin receptor-in skin secretions of amphibians.

Amphibian skin secretion represents a cerain scientific interest as a source of biologically active natural peptides. In the present research skin peptidome of wide-spread European frog Rana lessonae (Camerano, 1882) was studied for the first time ever. Peptide sequencing was accomplished with Fourier transform ion cyclotron resonance mass spectrometer in collision-induced and electron capture dissociation modes. A portion of amphibian peptides contains intramolecular C-terminal disulfide cycle which obstructs mass spectrometric sequencing. Two methods were utilized to overcome this difficulty: reduction with dithiotreithol followed by thiol group alkylation and oxidation into sulfonic acid groups with performic acid. Integrated approach employed in the present study allowed the identification of 49 peptides (of 6 to 37 amino acid residues), including 19 novel species.

Hyla arborea schelkownikowi is one of the leaf frog species inhabiting the southern territories of Russia and the former USSR. This frog species is a member of the Hylidae Rafinesque, 1815 batrachians family. The present study deals with the previously uninvestigated peptidome of the Hyla arborea schelkownikowi skin secretion. Nano-electrospray ionization Fourier transform mass spectrometry (nanoESI-FTMS) of the skin secretion, in the intact form and after acetylation, was selected as the general method of analysis. Electron-capture dissociation (ECD) and collision-induced dissociation (CID) fragmentation were both employed, while de novo sequencing was performed by manual interpretation of the MS data. The suppression of the cyclization of b-ions in the mass spectrometer by the acetylation reaction proved to be very efficient for the de novo sequencing of short peptides. Ten skin peptides were found and all of them, except for bradykinin, had not previously been reported. Six of the peptides belong to the tryptophyllins and related peptides, while three peptides are similar to the aureins.

Amphibian skin glands are known to secrete various types of bioactive peptides. The array of these peptides is specific for every frog species. The present research deals with the identification of peptides isolated from the skin secretion of the Marsh frog R. ridibunda inhabiting the Kolkhida Canyon of the Caucasian region. The research is based on comprehensive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of intact and chemically modified peptides. In particular, an oxidation procedure was applied directly to the crude skin secretion to open SS loops whereas N-terminal acetylation was additionally carried out for one individual peptide. Sequences were determined by manual interpretation of electron capture dissociation (ECD) and collisionally induced dissociation (CID) tandem mass spectra. A total of 29 peptides were identified in the skin secretion of the Caucasian Marsh frog. The peptide profile is represented with disulfide-containing peptides belonging to the brevinin, esculentin and ranatuerin families, neuropeptides of the bradykinin and bombesin families. Two identified peptides belonging to the ranatuerins are the first peptides of this family discovered in the skin secretions of European frogs. Ten of the identified peptides coincide with those reported earlier for the European Edible frog. Another ten are identical to those found in R. ridubunda from the Moscow region. This fact verifies the described method as being an efficient analytical tool to compare intra- and interspecific variabilities. Copyright © 2008 John Wiley & Sons, Ltd.

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC–MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.

Protein phosphorylation is a post-translational modification that is essential to cellular signaling, cellular function, and associated disease progression. Bottom-up proteomics based on enzymatic digestion is the most widely used approach for identifying and quantifying phosphoproteins in complex biological samples. Researchers have largely optimized the experimental conditions for trypsin digestion, and it is now a routine procedure. However, trypsin digestion is impaired by the presence of phosphorylated residues in the protein sequence. This impairment arises from the fact that there are commonly salt bridges between a negatively charged phosphate group and the side chain of protonated arginine or lysine. On average, 55% of all phosphopeptides have their phosphosites located less than three amino acid residues from a cleavage site. Salt bridges reduce the cleavage accessibility for trypsin by masking the basic site chain groups of arginine and lysine. Thus, there are frequent missed cleavages in the vicinity of phosphorylation sites, thereby lessening both the depth of proteome coverage and the quantification accuracy of phosphoproteomics. In this work, we propose a method termed PhosphoShield to mitigate salt bridge formation by adding a digallium complex that exhibits a high binding affinity to the phosphate group. We tested our method using quantitative mass spectrometry analysis of the phosphoproteome of human liver cancer cells (HepG2). PhosphoShield enhances the cleavage frequency of at least 17% of tryptic phosphopeptides having cleavage sites close to the phosphate group.