Label-free quantitative proteomic analysis reveals potential biomarkers for early healing in cutaneous leishmaniasis

Background Leishmaniasis is a parasitic disease caused by more than 20 species of the Leishmania genus. The disease is globally distributed and is endemic in 97 countries and three territories in the tropical and subtropical regions. The efficacy of the current treatments is becoming increasingly low either due to incomplete treatment or resistant parasites. Failure of treatment is frequent, and therefore, the search for early biomarkers of therapeutic response in cutaneous leishmaniasis (CL) is urgently needed. Objective The aim of this study was to compare the proteomic profiles in patients with CL before and after 7 days of treatment and identify early biomarkers of curative response. Methods Four patients with a parasitological diagnosis of leishmaniasis with confirmation of species by PCR-RFLP were recruited. All patients had a single lesion, and a protein from the middle of the ulcer was quantified by liquid chromatography and mass spectrometry. Results A total of 12 proteins showed differential expression in the comparative LC-electrospray ionization MS/MS (LC-ESI-MS/MS) triplicate analysis. Seven of them were up-regulated and five of them were down-regulated. Calcium binding proteins A2, A8, and A9 and hemoglobin subunits alpha-2 and delta showed high correlation with epidermis development and immune response. Conclusion We identified changes in the profiles of proteins that had a positive therapeutic response to the treatment. The proteins identified with differential expression are related to the reduction of inflammation and increased tissue repair. These proteins can be useful as biomarkers for early monitoring of therapeutic response in CL

Pronounced plastic and evolutionary responses to unpredictable thermal fluctuations in <i>Drosophila simulans</i>

Organisms are exposed to temperatures that vary, for example on diurnal and seasonal time scales. Thus, the ability to behaviorally and/or physiologically respond to variation in temperatures is a fundamental requirement for long-term persistence. Studies on thermal biology in ectotherms are typically performed under constant laboratory conditions, which differ markedly from the variation in temperature across time and space in nature. Here, we investigate evolutionary adaptation and environmentally induced plastic responses of Drosophila simulans to no fluctuations (constant), predictable fluctuations or unpredictable fluctuations in temperature. We whole-genome sequenced populations exposed to 20 generations of experimental evolution under the three thermal regimes and examined the proteome after short-term exposure to the same three regimes. We find that unpredictable fluctuations cause the strongest response at both genome and proteome levels. The loci showing evolutionary responses were generally unique to each thermal regime, but a minor overlap suggests either common laboratory adaptation or that some loci were involved in the adaptation to multiple thermal regimes. The evolutionary response, i.e., loci under selection, did not coincide with induced responses of the proteome. Thus, genes under selection in fluctuating thermal environments are distinct from genes important for the adaptive plastic response observed within a generation. This information is key to obtain a better understanding and prediction of the effects of future increases in both mean and variability of temperatures

Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings

Switchgrass (Panicum virgatum) is a perennial crop producing deep roots and thus highly tolerant to soil water deficit conditions. However, seedling establishment in the field is very susceptible to prolonged and periodic drought stress. In this study, a “sandwich” system simulating a gradual water deletion process was developed. Switchgrass seedlings were subjected to a 20-day gradual drought treatment process when soil water tension was increased to 0.05 MPa (moderate drought stress) and leaf physiological properties had expressed significant alteration. Drought-induced changes in leaf proteomes were identified using the isobaric tags for relative and absolute quantitation (iTRAQ) labeling method followed by nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) analysis. Additionally, total leaf proteins were processed using a combinatorial library of peptide ligands to enrich for lower abundance proteins. Both total proteins and those enriched samples were analyzed to increase the coverage of the quantitative proteomics analysis. A total of 7006 leaf proteins were identified, and 257 (4% of the leaf proteome) expressed a significant difference (p \u3c 0.05, fold change \u3c0.6 or \u3e1.7) from the non-treated control to drought-treated conditions. These proteins are involved in the regulation of transcription and translation, cell division, cell wall modification, phyto-hormone metabolism and signaling transduction pathways, and metabolic pathways of carbohydrates, amino acids, and fatty acids. A scheme of abscisic acid (ABA)-biosynthesis and ABA responsive signal transduction pathway was reconstructed using these drought-induced significant proteins, showing systemic regulation at protein level to deploy the respective mechanism. Results from this study, in addition to revealing molecular responses to drought stress, provide a large number of proteins (candidate genes) that can be employed to improve switchgrass seedling growth and establishment under soil drought conditions (Data are available via ProteomeXchange with identifier PXD004675)

A pathway proteomic profile of ischemic stroke survivors reveals innate immune dysfunction in association with mild symptoms of depression: a pilot study

Depression after stroke is a common occurrence, raising questions as to whether depression could be a long-term biological and immunological sequela of stroke. Early explanations for post-stroke depression (PSD) focused on the neuropsychological/psychosocial effects of stroke on mobility and quality of life. However, recent investigations have revealed imbalances of inflammatory cytokine levels in association with PSD, though to date, there is only one published proteomic pathway analysis testing this hypothesis. Thus, we examined the serum proteome of stroke patients (n = 44, mean age = 63.62 years) and correlated these with the Montgomery–Åsberg Depression Rating Scale (MADRS) scores at 3 months post-stroke. Overall, the patients presented with mild depression symptoms on the MADRS, M = 6.40 (SD = 7.42). A discovery approach utilizing label-free relative quantification was employed utilizing an LC-ESI–MS/MS coupled to a LTQ-Orbitrap Elite (Thermo-Scientific). Identified peptides were analyzed using the gene set enrichment approach on several different genomic databases that all indicated significant downregulation of the complement and coagulation systems with increasing MADRS scores. Complement and coagulation systems are traditionally thought to play a key role in the innate immune system and are established precursors to the adaptive immune system through pro-inflammatory cytokine signaling. Both systems are known to be globally affected after ischemic or hemorrhagic stroke. Thus, our results suggest that lowered complement expression in the periphery in conjunction with depressive symptoms post-stroke may be a biomarker for incomplete recovery of brain metabolic needs, homeostasis, and inflammation following ischemic stroke damage. Further proteomic investigations are now required to construct the temporal profile, leading from acute lesion damage to manifestation of depressive symptoms. Overall, the findings provide support for the involvement of inflammatory and immune mechanisms in PSD symptoms and further demonstrate the value and feasibility of the proteomic approach in stroke research

Comparison of two proteomic methods for analyses of glaucoma medication effects in vitro

Tämän syventävän työn tarkoituksena oli vertailla kahden eri massaspektrometrimenetelmän, SWATH-MS:n ja iTRAQ:n, eroja silmäsolujen proteomiikan tutkimuksissa. Tutkimustyö oli osa laajempaa kokonaisuutta, jossa tutkittiin glaukoomalääkkeiden vaikutusta silmän pinnan soluihin. Analysoitavina solumalleina käytettiin kahta silmän pinnan solulinjaa, HCE ja NHC. HCE-solulinja on kehitetty ihmisen sarveiskalvon epiteelisoluista, ja NHC-solulinja ihmisen sidekalvon soluista. Solut altistettiin kahdella eri glaukoomalääkkeellä, tafluprostilla ja latanoprostilla, sekä lääkkeiden säilöntäaineella bentsalkoniumkloridilla. Kontrollinäytteinä käytettiin altistamattomia soluja. Altistusten jälkeen kummankin solulinjan näytteet analysoitiin kahdella eri massaspektrometrimenetelmällä, SWATH-MS ja iTRAQ. Menetelmien välillä vertailtiin niiden tunnistamien proteiinien kokonaismäärää ja tunnistamisen toistettavuutta, pitoisuusmääritysten luotettavuutta ja tulosten yhteneväisyyttä keskenään. SWATH- MS:lla proteiineja tunnistettiin huomattavasti enemmän ja toistettavammin kuin iTRAQ:lla. iTRAQ vaikutti tulosten perusteella tuottavan luotettavampia pitoisuusmäärityksiä kuin SWATH-MS, mutta tulokset analysoitiin pienemmästä määrästä proteiineja. Pitoisuusmääritysten osalta tulokset olivat jokseenkin yhteneviä, mutta tuloksissa ei löytynyt lineaarista suhdetta menetelmien välillä

Global Acetylation Dynamics in the Heat Shock Response of Saccharomyces cerevisiae

All organisms face a constant barrage of environmental stresses. Single-cell organisms such as Saccharomyces cerevisiae, or common Baker’s yeast, must rely solely on cellular responses in order to survive. This response must occur in a rapid and highly coordinated manner to quickly inhibit all unnecessary processes and shuttle all available resources to those necessary for survival. One method that cells utilize for rapid protein regulation is the use of post-translational modifications. Enzymes within the cell add or remove a variety of chemical modifications, thus altering the local chemical environment of a protein. This creates a conformational change in the protein that can increase, decrease, or completely change the activity of the protein, as well as target them for relocation or degradation. Examples of common post-translational modifications include phosphorylation, ubiquitination, and the focus of this dissertation, acetylation. That protein acetylation occurs has been known for decades, but it is only recently that advances in technology such as high-resolution mass spectrometry and immunoprecipitation have led to the recognition of thousands of acetylated proteins across all domains of life. The roles and regulation of this modification, however, are still widely unknown. One approach to better understand possible roles for acetylation is to look at its dynamics in response to environmental stress. In this dissertation, I examine global changes in protein acetylation in the response of Saccharomyces cerevisiae to a mild heat shock and the potential mechanisms regulating these changes. Following an introductory literature review, this dissertation will cover the results of a large time-scale profiling of acetylome dynamics in response to heat shock. Proteins identified in this experiment are enriched for many cellular processes, suggesting that acetylation may play a much wider regulatory role than previously believed. These proteins are also enriched for interactions with many lysine acetyltransferases and deacetylases, suggesting that the regulation of this modification is complex. The next chapter will then discuss possible mechanisms regulating this response. This includes the investigation into concentrations of metabolites known to affect acetylation and deacetylation, lysine acetyltransferase and deacetylase complex remodeling, and localization changes for those complexes within the cell

Identificastion and quantification of metal-labeled peptides and proteins using matrix-assisted laser desorption/ionization mass spectrometry

Die Quantifizierung mit MeCAT (metal-coded affinity tags) bietet in der quantitativen Proteomanalyse eine vielversprechende Alternative z. B. zu Methoden wie ICAT oder iTRAQ, da hier sowohl differentielle Analysen möglich sind, aber auch die absolute Quantifizierung der jeweiligen Proteine mittels ICP-MS (Massenspektrometrie mit induktiv gekoppeltem Plasma) erfolgen kann. In der folgenden Arbeit wurde die erfolgreiche Identifizierung und Quantifizierung Metall-DOTA-markierter Peptide (Amino- und Sulfhydryl-gerichtete Markierung) für Peptide sowie Peptidmischungen mittels (LC-)MALDI MS und MS/MS gezeigt. Schließlich wurde die Lanthanoid-DOTA-Markierung auch zur Quantifizierung des tryptischen Abbaus von Neurotensin, sowie der Bildung der daraus entstehenden Spaltprodukte verwendet.MeCAT-labeling (metal-coded affinity tags) is an interesting new quantification approach compared to methods like ICAT or iTRAQ. It enables simultaneously differential but also absolute quantification of peptides via ICP-MS (inductively-coupled plasma mass spectrometry). In the following, the successful identification and quantification of metal-DOTA-labeled peptides (amino- and sulfhydryl-directed labeling) or peptide mixtures was shown using (LC-) MALDI MS and MS/MS. Finally lanthanoide-DOTA-labeling was applied to quantify the tryptic degradation of neurotensin and the formation of the reaction products