In-gel Trypsin Digestion Research Articles

Anti-inflammatory, remorin-like protein from green marine Macroalga Caulerpa sertularioides (S.G.Gmel.) M.Howe

The most prevalent natural source of hydrocolloids, cosmetics, medications, and nutraceuticals is marine seaweed (macroalgae). Numerous bioactivities, including antiviral, anticancer, anti-inflammatory, and immunomodulatory characteristics, have been found in bioactive substances such as polyphenols and sulfated and non-sulfated polysaccharides. As a result, new start-up projects and industries based on seaweed are emerging in all regions of the world with abundant marine biodiversity. In this current investigation, the anti-inflammatory activity of two different marine macroalgae Caulerpa racemosa (CR) and Caulerpa sertularioides (CS) was evaluated. Consequently, CS demonstrated more anti-inflammatory and antioxidant effects at a lower dose than CR. The IC50 value for DPPH inhibition was 456.1 μg/mL, and 180.9 μg/mL for CS and CR respectively. A similar result was obtained in the case of protein denaturation (PD), membrane stabilization (MS), and protease inhibition (PI) anti-inflammatory assays with 127.2 μg/mL, 135.5 μg/mL, and 71.88 μg/mL for CR, and 66.78 μg/mL, 88.96 μg/mL, and 59.54 μg/mL for CS respectively. Based on the SDS-PAGE, the molecular weight of lectin responsible for the anti-inflammatory activity was determined as 17 kDa. Protein mass fingerprinting was performed for the particular lectin by in-gel trypsin digestion, MALDI-MS analysis, and Mascot peptide mass fingerprinting. Because of this, the unidentified lectin protein was discovered to be a remorin-like protein that shared 65% of its sequence with the remorin-like protein of Aegilops tauschii subsp. tauschii. Therefore, it is the hitherto report on the presence of remorin-like protein from the green macroalga Caulerpa sertularioides.

Type I-like metalloproteinase in the venom of the West African saw-scaled carpet viper (Echis ocellatus) has anti-trypanosomal activity against African trypanosomes

African trypanosomiasis is an infectious disease caused by hemoparasites of the genus Trypanosoma and remains a major health problem in Africa - killing around 4000 people and animals worth an estimated $5 billion, annually. The absence of a vaccine and satisfactory drug against African trypanosomiasis (AT) necessitates the continued search for new chemotherapy options. Owing to the rich biochemical diversity in snake venom, it has recently become a source of therapeutic peptides that are being explored for the development of novel drug candidates for diverse ailments such as cancers and infectious diseases. To explore this, Echis ocellatus venom (EOV) was investigated for the presence of an anti-Trypanosoma factor, with the subsequent aim to isolate and identify it. Crude EOV was collected and tested in vitro on the bloodstream form (BSF) i.e. long and slender morphological form of Trypanosoma brucei and T. congolense. This initial testing was followed by a sequential anti-trypanosomal assay guided purification of EOV using ethanol precipitation, distillation, and ion exchange (IEX) chromatography to obtain the active trypanocidal component. The purified anti-Trypanosoma factor, estimated to be a 52-kDa protein on SDS-PAGE, was subjected to in-gel trypsin digestion and 2D RP HPLC-MS/MS to identify the protein. The anti-Trypanosoma factor was revealed to be a zinc-dependent metalloproteinase that contains the HEXXHXXGXXH adamalysin motif. This protein may provide a conceptual framework for the possible design of a safe and effective anti-trypanosomal peptide for the treatment of AT.

Organophosphorus Pesticides Promote Protein Cross-Linking.

Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 μg of MAP-rich tubulin with 100 μM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.

Alpha-amylase as the culprit in an occupational mealworm allergy case

BackgroundOccupational allergy has been described in employees working in contact with mealworms in pet stores, live fish bait or infested stored grains and recently, in mealworm farming for animal feed and human consumption. Mealworm allergens linked to occupational allergy are troponin C, cockroach-like allergen, tropomyosin, arginine kinase, early-staged encapsulation inducing- and larval cuticle proteins.ObjectiveWe report a case of occupational mealworm allergy and studied the culprit component.MethodsDiagnosis was done by skin prick, specific IgE, basophil activation and lung function testing. Allergen purification was performed by anion-exchange chromatography and immunoblotting with patient IgE. Allergens were identified by in-gel trypsin digest and tandem mass spectrometry. Allergenicity and specificity further confirmed by IgE inhibition and passive basophil activation experiments.ResultsWe describe a new case of occupational mealworm allergy in a laboratory worker, with sensitization to different developmental stages and derivates of the mealworm. In basophil activation tests, the majority of patient's basophils (69%–91%) degranulated upon stimulation with the lowest concentration of mealworm extracts (0.16 µg/ml). Despite strong sensitization to mites, the patient did not show cross-reactivity to other insects. We were able to identify alpha-amylase as the main allergen and through inhibition experiments, we demonstrated that low amounts (0.1 µg/ml) of this allergen could strongly inhibit mealworm specific IgE by 79.1%. Moreover, passive BAT experiments demonstrated the IgE-alpha-amylase interaction to be functional, inducing up to 25.5% degranulation in healthy donor basophils.ConclusionAlpha-amylase can be identified as the responsible allergen in this specific case of occupational mealworm allergy.

Proteomic Analysis of Human Breast Milk to Reveal Potential Protein Biomarkers for Breast Cancer

Breast cancer (BC), one of the most common cancers, is a leading cause of death for women in the United States. An estimated 1 in 8 women in the United States will develop BC in their lifetime. Early diagnosis and treatment of BC is crucial, and protein biomarkers for this disease could make this possible. Mass spectrometry (MS)‐based proteomic methods are ideal for the investigation of protein biomarkers. This study employs MS‐based proteomics to study the protein differences in human breast milk from women with BC and matched controls. If significant protein dysregulations are revealed, they could be considered potential future protein biomarkers of BC for diagnosis and treatment. In this study, six human breast milk samples were analyzed by performing a 2D‐SDS‐PAGE and further analyzed via nanoLC‐MS/MS. The human breast milk samples consisted of three comparison pairs of BC vs. control. There were one Coomassie blue‐stained gel (6 total gels) and two replicate silver‐stained gels (12 total gels) for each sample of breast milk. The silver‐stained gels were scanned using a laser densitometer and the images were analyzed using Progenesis Same Spots and Progenesis PG240 software. The spot percentages were measured for each dysregulated spot., An in‐gel trypsin digestion was performed for each spot with statistically significant dysregulation. This was followed by nanoLC‐MS/MS, data processing, database search and statistical analysis. The dysregulated proteins found in this study can be investigated as BC biomarkers for future clinical methods for early diagnosis and treatment of BC.

Analysis of the lake trout heart, blood, liver, and brain proteome using evolutionary proteomics

IntroductionSalvelinus namaycush (lake trout) is a top‐predator fish in the Great Lakes region. The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) use lake trout as a bioindicator of chemical stress by monitoring the concentrations of persistent, bioaccumulative and toxic (PBT) chemicals in their bodies. Studies have shown that elevated concentrations of PBTs can cause changes in transcribed genes, translated mRNAs, proteins produced, and post‐translational modifications in aquatic species. Though lake trout is used to monitor chemical concentrations for over 50 years, there is very little information available on the proteome of this species. A well‐developed protein database for the lake trout would help elucidate the effects of PBT chemicals on this species providing a direct health metric for the Great Lakes ecosystem.MethodsIn this study, heart, blood, brain, and liver samples from lake trout were analyzed by SDS‐PAGE, followed by in‐gel trypsin digestion and analysis by nanoLC‐MS/MS. The raw data was searched against different NCBI and UniProtKB databases in Mascot Daemon and the output was analyzed by Scaffold 4.3 software. Databases used include Actinopterygii, Salmonidae, Salvelinus, as well as the highly studied species Oncorhynchus mykissand Danio rerio.Preliminary DataPreviously, our group has reported a large number of proteins for lake trout liver, heart and blood (Dupree et al., Proteomics, 2019, PMID: 31578773 and Dupree et al., Proteomics, 2021, PMID: 34676671). In lake trout liver we identified 4194 proteins using the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the blood and heart we identified 838 and 580 proteins, respectively, using NCBI databases and 1180 and 561 potential protein hits for the heart and blood, respectively in BR2. A lake trout brain proteomics analysis completed using an in‐house SDS‐PAGE, followed by in gel digestion and nanoLC‐MS/MS analysis. The Mascot database search is underway. Once protein hits are cataloged, BLAST and Scaffold‐based comparisons of the liver, heart, blood and brain will be performed. Additional search of the data against the other fish relatives will also be completed. Through this process, evolutionary relationships for the lake trout species will also be explored. The current study will add to our understanding of the lake trout proteome that will be used to understand the biochemical/physiological effects of legacy PBT chemicals in the Great Lakes ecosystem.Novel AspectUse of four types of lake trout tissue together to develop a protein database for the species.

Investigating Toxin Diversity and Abundance in Snake Venom Proteomes.

Understanding snake venom proteomes is becoming increasingly important to understand snake venom biology, evolution and especially clinical effects of venoms and approaches to antivenom development. To explore the current state of snake venom proteomics and transcriptomics we investigated venom proteomic methods, associations between methodological and biological variability and the diversity and abundance of protein families. We reviewed available studies on snake venom proteomes from September 2017 to April 2021. This included 81 studies characterising venom proteomes of 79 snake species, providing data on relative toxin abundance for 70 species and toxin diversity (number of different toxins) for 37 species. Methodologies utilised in these studies were summarised and compared. Several comparative studies showed that preliminary decomplexation of crude venom by chromatography leads to increased protein identification, as does the use of transcriptomics. Combining different methodological strategies in venomic approaches appears to maximize proteome coverage. 48% of studies used the RP-HPLC →1D SDS-PAGE →in-gel trypsin digestion → ESI -LC-MS/MS pathway. Protein quantification by MS1-based spectral intensity was used twice as commonly as MS2-based spectral counting (33–15 studies). Total toxin diversity was 25–225 toxins/species, with a median of 48. The relative mean abundance of the four dominant protein families was for elapids; 3FTx–52%, PLA2–27%, SVMP–2.8%, and SVSP–0.1%, and for vipers: 3FTx–0.5%, PLA2–24%, SVMP–27%, and SVSP–12%. Viper venoms were compositionally more complex than elapid venoms in terms of number of protein families making up most of the venom, in contrast, elapid venoms were made up of fewer, but more toxin diverse, protein families. No relationship was observed between relative toxin diversity and abundance. For equivalent comparisons to be made between studies, there is a need to clarify the differences between methodological approaches and for acceptance of a standardised protein classification, nomenclature and reporting procedure. Correctly measuring and comparing toxin diversity and abundance is essential for understanding biological, clinical and evolutionary implications of snake venom composition.

Proteomics and phosphoproteomics of C3 to CAM transition in the common ice plant.

Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C3 to CAM transition in the common ice plant (Mesembryanthemum crystallinum). Overall, our workflow provides robust methods for the identification of phosphoproteins and total proteins. It can be broadly applied to many other organisms and sample types, and the results provide a more accurate picture of the molecular switches that regulate different biological processes.

Plant secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation.

Plant cell walls are typically composed of polysaccharide polymers and cell wall proteins (CWPs). CWPs account for approximately 10% of the plant cell wall structure and perform a wide range of functions. Previous studies have identified approximately 1000 CWPs in the model plant Arabidopsis thaliana; however, the analyses mainly targeted primary cell walls, which are generated at cell division. In contrast, little is known about CWPs in secondary cell walls (SCWs), which are rigid and contain the phenolic polymer lignin. Here, we performed a cell wall proteome analysis to obtain novel insights into CWPs in SCWs. To this end, we tested multiple methods for cell wall extraction with cultured Arabidopsis cells carrying the VND7-VP16-GR system, with which cells can be transdifferentiated into xylem-vessel-like cells with lignified SCWs by dexamethasone treatment. We then subjected the protein samples to in-gel trypsin digestion followed by LC-MS/MS analysis. The different extraction methods resulted in the detection of different cell wall fraction proteins (CWFPs). In particular, centrifugation conditions had a strong impact on the extracted CWFP species, resulting in the increased number of identified CWFPs. We successfully identified 896 proteins as CWFPs in total, including proteases, expansins, purple phosphatase, well-known lignin-related enzymes (laccase and peroxidase), and 683 of 896 proteins were newly identified CWFPs. These results demonstrate the usefulness of our CWP analysis method. Further analyses of SCW-related CWPs could be expected to produce information useful for understanding the roles of CWPs in plant cell functions.

A new thermostable rhizopuspepsin: Purification and biochemical characterisation

The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25−65 % saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 °C, respectively. The enzyme was unusually thermostable, as it retained 50 % of its activity beyond 85 °C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63 %) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with “experimental evidence at protein-level”.

Classic Pentachlorophenol Hydroxylating Phenylalanine 4-Monooxygenase from Indigenous Bacillus tropicus Strain AOA-CPS1: Cloning, Overexpression, Purification, Characterization and Structural Homology Modelling.

The metabolically promiscuous pentachlorophenol (PCP) hydroxylating Phe4MO (represented as CpsB) was detected, amplified (from the genome of Bacillus tropicus strain AOA-CPS1), cloned, overexpressed, purified and characterized here. The 1.755-kb gene cloned in the pET15b vector expressed a ≅ 64kDa monomeric protein which was purified to homogeneity by single-step affinity chromatography, with a total yield of 82.1%. The optimum temperature and pH of the enzyme were found to be 30°C and 7.0, respectively. CpsB showed functional stability between pH 6.0-7.5 and temperature 25-30°C. The enzyme-substrate reaction kinetic studies showed the allosteric nature of the enzyme and followed pre-steady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465μM.s-1, 140μM, 0.099s-1 and 7.07 × 10-4μM-1.s-1, respectively, for the substrate PCP. The in-gel trypsin digestion experiments and bioinformatic tools confirmed that the reported enzyme is a Phe4MO with multiple putative conserved domains and metal ion-binding site. Though Phe4MO has been reported to have a diverse catalytic function, here we report, for the first time, that it functions as a PCP dehalogenase or PCP-4-monooxygenase by hydroxylating PCP. Hence, the use of this enzyme may be further explored in the bioremediation of PCP and other related xenobiotics.

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.

Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities. A novel thiosulfate:quinone oxidoreductase from strain SH (SH-TQO) has been purified from its solubilized membrane fraction. The gene for SH-TQO was determined from the draft genome sequence of the strain SH. Amino acid sequences of peptides generated by the in-gel trypsin digestion of SH-TQO were found in a protein encoded by locus tag B1757_09800 of the genome of the strain SH. The gene encoded 444 amino acids with a signal peptide of 29 amino acids and was annotated to encode a porin. The gene was located in a unique genomic region, not found in A. thiooxidans strains, suggesting that the strain SH acquired this region through a horizontal gene transfer. A protein–protein basic local alignment search revealed that sulfur-oxidizing bacteria, such as Acidithiobacillus species have proteins homologous to SH-TQO, though the degree of homologies was relatively low. The protein, DoxXA, which is homologous to TQO from Acidianus amvibalens, was also found in the genomic region.

A method combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem-mass spectrometry to detect circulating immune complexes between therapeutic monoclonal antibodies and anti-drug antibodies in animals

Therapeutic monoclonal antibodies can potentially induce unwanted immune responses, resulting in the production of anti-drug antibodies (ADAs). The binding of ADAs to drugs and subsequent formation of immune complexes (ICs) can trigger various responses, dependent on the size, concentration, and subclass of ADAs. To better understand the impact of ADAs on pharmacokinetics, pharmacodynamics, and toxicological profiles, a bioanalytical method was developed for the detection of ICs between human monoclonal immunoglobulin G (IgG) and ADAs in biological samples. Regarding the experimental procedure, in brief, the human antibody-specific ICs and unbound human antibody in biological samples are separated through blue native polyacrylamide gel electrophoresis (BN-PAGE). The target fractions are then cut from the gel, followed by in-gel trypsin digestion and subsequent liquid chromatography tandem-mass spectrometry (LC-MS/MS) to monitor the human IgG-specific peptide. This method was able to detect various types of human antibodies with a lower limit of detection of 10 μg/mL in monkey serum. The assay performance for the detection of ICs was demonstrated using spiked samples, and pre-incubated ICs in monkey serum were clearly detected. Taken together, these findings indicate that our method enables a semi-quantitative analysis for estimating the ratio of human antibody included ICs in comparison to the total antibody. This method was successfully applied to an in vivo study using mice, and the data helped explain the unexpectedly rapid clearance of a humanized antibody due to the formation of large ICs. The combination of the separation of ICs by BN-PAGE and the detection of the human IgG-specific peptide by LC-MS/MS is a useful general bioanalytical approach for the detection of ICs in animals.

Purification and characterizations of a nanocage ferritin GF1 from oyster (Crassostrea gigas)

Ferritin can form nanocage architectures and has demonstrated potential to serve as functional nanomaterials. Here, the oyster (Crassostrea gigas) ferritin gene GF1 was obtained from the viscera of the oyster, successfully cloned, prokaryotically expressed in E. coli BL21 (DE3), purified and characterized. Oyster ferritin was purified by ion exchange chromatography using DEAE-Sepharose Fast Flow. The molecular weight of the subunit was 19972 Da (polyacrylamide gel electrophoresis), and the oyster ferritin weight was 480 kDa (non-denaturing gel electrophoresis). After in-gel trypsin digestion, ferritin was identified using NanoLC-Q-TOF-MS and then analyzed by circular dichroism (CD) spectrometry for its secondary structure. Ferritin nanocages were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The three-dimensional structure of oyster ferritin was constructed based on the amino acid sequence of human ferritin heavy chain by the homology modeling method. Here, we provide a theoretical foundation for ferritin self-assembly and biological functional activity.

Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct Conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (∗ indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

Proteome Analysis of 14-3-3 Targets in Tomato Fruit Tissues.

Tomato is a major crop plant and an important constituent of the human diet. Exclusive features such as bearing fleshy fruits and undergoing a phase transition from partially photosynthetic to fully heterotrophic metabolism make tomato fruit a model system for fruit development studies. Although the tomato genome has been completely sequenced, functional proteomics studies are still at their starting stage. Proteomics technologies, especially the combination of multiple approaches, provide a very powerful tool to accurately identify functional proteins and investigate certain sets of proteins in more detail. The direct binding of plant 14-3-3 proteins to their multiple target proteins modulates the functions of the latter, suggesting that these 14-3-3 proteins are directly involved in various physiological pathways. This chapter outline methods for the identification of 14-3-3 protein complexes in tomato fruit tissues. These methods include detailed protocols for protein extraction, coimmunoprecipitation, SDS-PAGE, SYPRO Ruby staining, in-gel trypsin digestion, and LC-MS/MS analysis for 14-3-3 interactomics.

AGE-RELATED CHANGES IN THE MUSCLE SECRETOME

Skeletal muscle is one of the most abundant tissues in the body. In addition to its key roles in body support, movement and metabolic homeostasis, muscle also functions as an endocrine/secretory organ producing and releasing proteins into the circulation that modulate distant tissues (i.e. myokines). Considering that muscle mass and function changes with advancing age, here we tested the hypothesis that aging alters the muscle secretome profile. After euthanasia, soleus muscles from sedentary young and old mice were dissected, and incubated in oxygenated KRB buffer for 2 h. The buffer was subjected to in-gel trypsin-digestion and peptides analyzed by mass spectrometry. The concentration of 36 proteins were significantly (P<0.05) elevated in the young vs. the old group. In contrast, only 7 proteins were significantly elevated in the old group. Some notable differences include those in HSPA1B and HSPA5 that were detected only in the young group. HSPA8 also was significantly elevated by 1.8-fold (P<0.05) in the young versus the old group. Another prominent difference between groups involved translationally controlled tumor protein (TCTP), a critical regulator of apoptosis/carcinogenesis, that was elevated by 7-fold in the young vs. the old group (P<0.05). These results indicate that aging alters the muscle secretion profile. Identified differences in the muscle secretome could reflect intrinsic changes in muscle cells with age. Because these myokines are released into the circulation, it is also possible that myokine secretion is a regulated cellular process by which muscle communicates and modulates the aging process in distant tissues.

Sweet Pepper (Capsicum annuum L.) Fruits Contain an Atypical Peroxisomal Catalase That is Modulated by Reactive Oxygen and Nitrogen Species.

During the ripening of sweet pepper (Capsicum annuum L.) fruits, in a genetically controlled scenario, enormous metabolic changes occur that affect the physiology of most cell compartments. Peroxisomal catalase gene expression decreases after pepper fruit ripening, while the enzyme is also susceptible to undergo post-translational modifications (nitration, S-nitrosation, and oxidation) promoted by reactive oxygen and nitrogen species (ROS/RNS). Unlike most plant catalases, the pepper fruit enzyme acts as a homodimer, with an atypical native molecular mass of 125 to 135 kDa and an isoelectric point of 7.4, which is higher than that of most plant catalases. These data suggest that ROS/RNS could be essential to modulate the role of catalase in maintaining basic cellular peroxisomal functions during pepper fruit ripening when nitro-oxidative stress occurs. Using catalase from bovine liver as a model and biotin-switch labeling, in-gel trypsin digestion, and nanoliquid chromatography coupled with mass spectrometry, it was found that Cys377 from the bovine enzyme could potentially undergo S-nitrosation. To our knowledge, this is the first report of a cysteine residue from catalase that can be post-translationally modified by S-nitrosation, which makes it especially important to find the target points where the enzyme can be modulated under either physiological or adverse conditions.

Abstract 3741: Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture

Abstract Cells respond to ionizing radiation (IR) through the activities of constitutively expressed proteins and through changes in gene expression. Constitutively expressed proteins play well-defined roles in major components of the cellular radioresponse, such as DNA repair and cell cycle. However, the contribution of changes in gene expression to the radioresponse is less understood. We have previously demonstrated through polysome profiling and microarray analysis that translational control is a key component of radiation-induced changes in gene expression. In response to IR, specific transcripts are recruited to or away from polysomes without affecting the global polysome profile of the cells. One mechanistic explanation for this observation is that IR influences the activity of RNA binding proteins (RBPs), which regulate the inclusion or exclusion of transcripts from polysomes. Here, we utilized RNA interactome capture (RIC) to identify RBPs bound to transcripts in control and irradiated human cancer cells. Cells were either mock irradiated or treated with 2Gy. One hour post-IR, RNA and protein complexes were crosslinked with 254 nm UV light. mRNA was isolated from cell lysates with oligo(dT) magnetic beads followed by several rounds of highly stringent washes to remove non-specific interactors. RNA was removed from crosslinked proteins by RNase digestion. The proteins were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, and the subsequent peptides were analyzed by label-free quantification mass spectrometry using an Orbitrap Fusion mass spectrometer. Proteome Discoverer 2.2 was used to search the data against human proteins from the UniProt database using SequestHT with a 1% false discovery rate. We found that we were able to efficiently pull down RNA:protein complexes with a crosslinking dose of 150 mJ/cm2. As determined by silver staining, the protein pattern of crosslinked samples differed greatly from protein isolated from whole cell lysates. By western blot, this method highly enriched for known RBPs, such as PTBP1 and CUGBP1, while contamination from non-RBPs, such as actin and tubulin, was not detected. By mass spectrometry, we identified hundreds of RBPs in each replicate above the no crosslinking background control for both untreated and irradiated cells. However, more RBPs were consistently detected in the control cells. Examples of RBPs preferentially binding in control cells include SSB, a protein involved in a variety of RNA metabolic processes, and U2AF2, a protein involved in RNA splicing. Overall, these data demonstrate that RIC is a sensitive and rigorous method to identify RBPs and that RBPs may play a role in radiation-induced post-transcriptional gene regulation. Preferential binders identified by this method may provide further insight into pathways regulating tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Theresa Wechsler, Gaelyn C. Lyons, Lisa M. Jenkins, Kevin Camphausen, Philip J. Tofilon. Identification of RNA binding proteins influenced by ionizing radiation through RNA interactome capture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3741.