The hyporheic zone (HZ) is a sensitive ecotone in river ecosystems because of its biodiversity and susceptibility to human activities. Hyporheic fauna are exposed to multiple stressors that affect ...the physiology and metabolism of organisms and ultimately ecosystem functioning and biodiversity. This study aimed to quantify adenine nucleotides in the stygophilous freshwater amphipod Synurella ambulans and to evaluate the potential of adenylate energy charge (AEC) as a physiological biomarker of general stress in the HZ of the Sava River at sites with different intensities of environmental and anthropogenic stress. Field studies were conducted seasonally (in December 2018 and April, July, and October 2019) at two sampling sites, one upstream (1-UP) and the other downstream (2-DOWN) of the discharge from the wastewater treatment plant using the standard piston pump. The amphipod population from site 1-UP had significantly higher AEC values than the population from site 2-DOWN in all seasons except summer. Coordinated changes in AEC values with the ATP/ADP ratio indicated differences in energy status between the two populations. However, no changes in the apparent equilibrium constant of adenylate kinase were observed, except in spring at site 1-UP. Multiple linear regression models showed the strongest associations of AEC with Fe and Zn accumulated in amphipods, followed by environmental factors (conductivity, dissolved O2, and concentrations of nitrites and phosphates in the interstitial water). AEC was shown to be a useful index of environmental stress in S. ambulans because it can directly measure the change in available energy and thus the metabolic stress to which the organism is exposed. Finally, seasonal and spatial variations in AEC values reflected ecological status in the HZ.
The starch availability and nutritional value of corn (Zea mays L.) are affected by zein proteins. The aim of the study was to see whether the proposed reduction of γ-zeins during the fermentation of ...silages is a result of either the enzymatic proteolytic activity or of the acidic environment, and how this reduction affects starch availability and degradability in high-moisture corn. A mass spectrometry (MS) technique was used to quantify the 16- and 27-kDa γ-zeins. Briefly, two-dimensional gel electrophoresis (2-DE) was used for γ-zein separation, followed by densitometry for protein quantification and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/TOF) for protein identification. The results show that the reduction in γ-zeins induced by the ensiling led to a more pronounced starch availability and in vitro degradation, and this reduction was dependent on the type of proteolysis. More specifically, the results indicate that the reduction of γ-zeins in the ensiled corn was primarily driven by the enzymatic proteolysis. Furthermore, we demonstrated that 2-DE followed by densitometric quantification and the mass spectrometry analysis for protein identification can be used as a state-of-the-art method for γ-zein evaluation both in fresh and fermented/ensiled corn samples.
First Proteome Analysis of Poplar-Type Propolis Šuran, Jelena; Radić, Božo; Trevisan-Silva, Dilza ...
Plant foods for human nutrition (Dordrecht),
03/2024, Letnik:
79, Številka:
1
Journal Article
Recenzirano
Propolis is a natural mixture of honeybee-released and plant-derived compounds produced by honeybees. Poplar propolis is rich in bioactive polyphenolic compounds, and due to its many health benefits, ...it is commonly used as a food supplement or functional food ingredient. However, it is the only honeybee product whose proteome hasn’t been analyzed. Here, we report a first proteome analysis of poplar-type propolis, a challenging glue-type resinous sample for protein characterization. Raw propolis mixture was precipitated with cold acetone to obtain the protein fraction. Proteins were digested with trypsin, and generated peptides were analyzed on nano-ESI-qTOF SYNAPT G2-Si mass spectrometer (MS) by data-independent acquisition (DIA) and data-dependent acquisition (DDA). Identified peptides and inferred proteins suggest the presence of new bioactive molecules as components of propolis. The poplar-type propolis proteome is composed of a mixture of proteins from the
Apis
and
Populus
genera. This is the first-ever report of the proteome of any type of propolis.
The process of identifying the protein targets and off-targets of a biologically active compound is of great importance in modern drug discovery. Various chemical proteomics approaches have been ...established for this purpose. To compare the different approaches, and to understand which method would provide the best results, we have chosen the EGFR inhibitor lapatinib as an example molecule. Lapatinib derivatives were designed using linkers with motifs, including amino (amidation), alkyne (click chemistry) and the diazirine group (photo-affinity). These modified lapatinib analogues were validated for their ability to inhibit EGFR activity in vitro and were shown to pull down purified recombinant EGFR protein. In all of the approaches evaluated here, we identified EGFR as the main protein target from the lysate of immortalised cell line expressing EGFR, thus validating its potential use to identify unknown protein targets. Taken together, the results reported here give insight into the cellular activities of lapatinib.
Metabolic syndrome, characterized by obesity, low-grade inflammation, insulin resistance, hyperglycemia, dyslipidemia and hypertension, is a major risk factor for cardiovascular mortality. ...Preclinical studies on recently discovered classes of lipids – fatty acid esters of hydroxy fatty acids (FAHFA) have revealed their anti-inflammatory and insulin-sensitizing potential. The FAHFA levels are significantly decreased in insulin-resistant individuals, their application exhibited anti-inflammatory effects and restoring the glucose-insulin homeostasis. The aim of our research was to analyze the overall FAHFA composition in a common diet, as only a partial FAHFA composition has been revealed so far (only the PAHSA subclass was analyzed in a few foods). A new approach to the FAHFAs analysis includes nano-LC and post-column modifier followed by negative ion mass spectrometry, in order to obtain maximum sensitivity. Analysis of different foods – oat (whole grain, coarse flakes and fine flakes), apple, clementine, lemon, strawberry, blueberry, mango, kiwi, avocado, pineapple, banana, onion, garlic, cherry tomato, carrot, parsley root, pepper and radish – exhibited wide inter-food variation in the FAHFA profiles. Sixteen analyzed FAHFAs (palmitic, oleic, palmitoleic and stearic hydroxy-esters) showed microgram to low nanogram levels (0.165 ng/g – 32 μg/g FW), with the highest abundancy in oat, clementine, garlic and pineapple. Stearic acid hydroxy stearic acid (SAHSA) was the most abundant FAHFA, especially in the food with antioxidative, anti-inflammatory and beneficial metabolic effects. In contrary, the PAHSA - previously proven to have the strongest antihyperglycemic and insulin-sensitizing effects, was not present in some foods (radish, avocado, mango, lemon, cherry tomato, kiwi). Our study proves the importance of overall FAHFA analysis in food (especially in a functional food), because of their potential metabolic benefits and possible future incorporation in special diets.
For mass spectrometry-based diagnostics of microorganisms, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used to identify urinary ...tract pathogens. However, it requires a lengthy culture step for accurate pathogen identification, and is limited by a relatively small number of available species in peptide spectral libraries (≤3329). Here, we propose a method for pathogen identification that overcomes the above limitations, and utilizes the MALDI-TOF/TOF MS instrument. Tandem mass spectra of the analyzed peptides were obtained by chemically activated fragmentation, which allowed mass spectrometry analysis in negative and positive ion modes. Peptide sequences were elucidated de novo, and aligned with the non-redundant National Center for Biotechnology Information Reference Sequence Database (NCBInr). For data analysis, we developed a custom program package that predicted peptide sequences from the negative and positive MS/MS spectra. The main advantage of this method over a conventional MALDI-TOF MS peptide analysis is identification in less than 24 h without a cultivation step. Compared to the limited identification with peptide spectra libraries, the NCBI database derived from genome sequencing currently contains 20,917 bacterial species, and is constantly expanding. This paper presents an accurate method that is used to identify pathogens grown on agar plates, and those isolated directly from urine samples, with high accuracy.
Our research objective was to investigate the use of 4'-formylbenzo-18-crown-6-ether (4fb18C6) for the covalent labelling of peptides for unambiguous peptide identification. Specifically, 23 peptides ...were analysed using a coupled system of liquid chromatography and tandem mass spectrometry with electrospray ionization to test de novo sequencing of derivatized and intact peptides. The reaction was optimized for reductive amination to be performed in an aqueous medium at pH 6 using the microwave radiation. After matching the tandem mass spectra of derivatized and intact peptides in the range of + or -0.005 Da, the chemical noise was reduced by up to 90% and six proteins from 17 different species were identified using the BLASTp algorithm (NCBInr and UniProtKB/Swiss-Prot databases). Species identification enabled further accelerated database search using the classical method of mass spectra database matching. The presented method enables reliable de novo sequencing of peptides and represents a new tool for species identification. Keywords: de novo sequencing, crown ether, N-terminal peptide derivatization, noise reduction, database matching, species identification.
Since silver nanoparticles (AgNPs) are a dominant nanomaterial in consumer products, there is growing concern about their impact on the environment. Although numerous studies on the effects of AgNPs ...on living organisms have been conducted, the interaction of AgNPs with plants has not been fully clarified. To reveal the plant mechanisms activated after exposure to AgNPs and to differentiate between effects specific to nanoparticles and ionic silver, we investigated the physiological, ultrastructural and proteomic changes in seedlings of tobacco (Nicotiana tabacum) exposed to commercial AgNPs and ionic silver (AgNO3) from the seed stage. A higher Ag content was measured in seedlings exposed to AgNPs than in those exposed to the same concentration of AgNO3. However, the results on oxidative stress parameters obtained revealed that, in general, higher toxicity was recorded in AgNO3-treated seedlings than in those exposed to nanosilver. Ultrastructural analysis of root cells confirmed the presence of silver in the form of nanoparticles, which may explain the lower toxicity of AgNPs. However, the ultrastructural changes of chloroplasts as well as proteomic study showed that both AgNPs and AgNO3 can affect photosynthesis. Moreover, the majority of the proteins involved in the primary metabolism were up-regulated after both types of treatments, indicating that enhanced energy production, which can be used to reinforce defensive mechanisms, enables plants to cope with silver-induced toxicity.
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•Higher accumulation of Ag was recorded after exposure to AgNPs compared to AgNO3.•Direct AgNPs uptake by root cells was confirmed.•AgNPs induced less severe oxidative damage than AgNO3.•Ultrastructural and proteome study showed that AgNPs and AgNO3 affect photosynthesis.•Primary metabolism proteins were up-regulated after both types of treatments.
Rationale
Selective derivatization of peptide N‐terminus with 4‐formyl‐benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge ...reduction conditions. Overlapped positive and negative tandem mass spectra show b‐ions making the assignment of b‐ion series fragments easy and accurate.
Methods
We developed an FBSA–peptide microwave‐assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non‐tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high‐quality data set of sulfonated b‐ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b‐ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y‐ions in positive tandem mass spectra to identify complete peptide sequences.
Results
The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high‐intensity signals of b‐ and y‐ions) compared to commonly used N‐terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b‐ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive‐ and negative ion modes, respectively. High visibility of b‐ion series in negative ion mode can be attributed to N‐terminal sulfonation that had no negative effect on the production of b‐ and y‐ion series in positive ion mode.
Conclusions
The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b‐ions in positive‐ and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.
Given the increasing need to protect vulnerable freshwater ecosystems and make them more resilient to human use and climate change, biomonitoring of the hyporheic zone (HZ), which plays a critical ...role in pollution attenuation, is essential. The aim of the present study was to assess the potential of the amphipod species Synurella ambulans as a bioindicator of metal contamination in the HZ of the Sava River (Croatia). Amphipods were collected during the four seasons at two sampling sites (average sampling depth 55 cm) differing in type (agricultural and urban) and intensity (diffuse and point source contamination) of anthropogenic influence, one located upstream (Medsave), and the other downstream (Jarun) of the wastewater treatment plant discharge. Concentrations of Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Rb, Sn, Zn, Ca, K, Mg and Na were measured in the interstitial water, sediments and specimens of S. ambulans by HR ICP-MS. Physicochemical parameters (temperature, DO, O2 saturation, pH, conductivity, alkalinity, total water hardness, CODKMnO4, nutrients) were measured in the interstitial water, while organic carbon was measured in the interstitial water and sediments. Metal concentrations in interstitial water and sediments were below thresholds set by environmental quality standards. Metal concentrations in S. ambulans were classified as follows: higher at the Jarun site (Al, Cr, Fe, Ni, Pb, Sn), higher at the Medsave site (Cd, Cu, Rb) and mostly comparable at both sites (Co, Mn, Zn). Bioaccumulation factors were generally higher at Jarun, with average values ranging from 322 to 143,278 L kg−1. Bioaccumulation of metals in S. ambulans depended on various environmental factors, with metal exposure level and dissolved macro elements showing the strongest association with metals accumulated in S. ambulans. The findings provided the first evidence on the suitability of S. ambulans as a good bioindicator of chronic metal contamination in the HZ.
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•Synurella ambulans was sampled seasonally in hyporheic zone (HZ) in agricultural and urban area.•Metal concentrations in S. ambulans showed various spatiotemporal patterns.•Metal concentrations in S. ambulans strongly correlated with exposure levels in water.•Metals accumulated in S. ambulans strongly associated with dissolved macro elements.•S. ambulans is suitable for biomonitoring of chronic metal contamination in HZ.
