Protease Inhibitor Cocktail Research Articles

Investigating the effect of linker peptides on the fragmentation of Fc-fusion proteins in transient gene expression of mammalian cells

Protein fragmentation is a critical quality attribute for Fc-fusion protein production in mammalian cells. In the production of viral non-structural proteins as the form of Fc-fusion protein, fragmentation of Fc-fusion proteins occurred in two transient gene expression (TGE) systems with human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. The introduction of a flexible empirical linker reduced fragmentation in HEK293 cells, but not in CHO cells. Additionally, two rigid empirical linkers failed to restore impaired Fc-fusion proteins in CHO cells. In vitro incubation assay using conditioned culture medium and cultures supplemented with protease inhibitor cocktail suggest that fragmentation of Fc-fusion proteins in CHO cells may be due to various host cell proteins released into the culture medium. These findings suggest that the introduction of linker peptides can improve the fragmentation of Fc-fusion proteins in mammalian cells, but exhibit different fragment patterns depending on the cell type.

Electrophoretic analysis of proteins from raw materials and commercial diets used in animal feed

In aquaculture, balanced feed accounts for 50% to 70% of the operational costs of cultivating aquatic organisms. This feed is made from various raw materials that serve as protein sources, which significantly influence the final cost of the feed. Therefore, an electrophoretic study was conducted to compare the protein content in different raw materials and commercial diets used in animal feed. Electrophoretic profiles with standard concentration gels (SDS-PAGE) were used to separate protein fractions from animal-origin raw materials, including Earthworm Meals of Eisenia andrei (HLF) and Eisenia fetida (HLT), meat-bone meal (MBM), and fish meal (FM), as well as plant-origin meals, such as Leucaena leaf meal (LLM), corn meal (YCM), and wheat bran (WBM). Commercial diets (A, B, and C) were also evaluated. The protein content of the samples was determined. For electrophoretic analysis, a protease inhibitor cocktail was used, along with a molecular weight standard with a range from 250 to 4 kDa. The molecular weights of the diets ranged between 31.1 and 234.1 kDa, 36.3 and 190.4 kDa, and 4.281 and 95.685 kDa. Some of the protein bands in the analyzed samples (HLF, HLT, FM, YCM, and WBM) were very close to the reference protein bands at 148, 98, 64, 50, and 36 kDa, corresponding to the molecular weight standard used. Electrophoretic characterization provided insight into the different protein fractions, offering valuable information about their potential functional and nutritional properties. Electrophoresis is an essential analytical tool for assessing both the quality and safety of raw materials and foods.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aβ42 and Aβ40 across all approaches. However, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Increasing protein identifications in bottom-up proteomics of T. castaneum − Exploiting synergies of protein biochemistry and bioinformatics

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant.Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications.A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant’s Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis.Our work with Tribolium castaneum larvae demonstrates that sometimes – depending on matrix and research question − more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Preparation and extraction of chorion proteins from Salmo salar embryos at the pigmented eye stage for electrophoresis with SDS-polyacrylamide gel

The chorion fulfills important functions in fish embryos, including protecting the embryo during development. The characterization of the protein profile of this envelope could be used as a bioindicator in the evaluation of the quality of embryonic development. The object of this work was to validate a standardized protocol for protein extraction from chorion of Salmo salar embryos at 280 accumulated thermal units (ATU) by comparing and combining existing methods. The protocol consists of consecutive washing of the chorion samples followed by protein extraction with the solution that was named SDS solution (Tris–HCl 100 mM (pH 8), Urea 8 M, 1% SDS, β-mercaptoethanol 300 mM and EGTA 10 Mm, and 1% protease inhibitor cocktail) and mechanical methods. Protein extraction is enhanced by a working temperature of 75 °C and use of a disperser. The protein concentration was quantified by Bradford Assay. After extraction, the samples were diluted (dilution factor 10) before reading against the calibration curve. After gel electrophoresis with a load of 3 µg of protein, staining showed more than 4 bands, with molecular weights between 25 kDa and 180 kDa.•The protein profile of fish chorion was between 25 kDa and 180 kDa.•Solution containing 1% SDS allows a higher extraction of proteins from the chorion of Atlantic salmon embryos with 280 ATU.•Chorion protein identification is a valuable tool in determining gamete and embryo quality in fish.

ОЦЕНКА ЛОКАЛЬНОГО ПРОФИЛЯ ЦИТОКИНОВ, ЭКСПРЕССИРУЕМЫХ НАТИВНЫМИ КЛАПАНАМИ СЕРДЦА, ПОЛУЧЕННЫМИ ОТ ПАЦИЕНТОВ С ПРИОБРЕТЕННЫМИ ПОРОКАМИ СЕРДЦА

Doing. The pathogenesis of acquired heart defects is a complex process involving many different factors. One of the triggers for the development of dysfunctions of native heart valves can be an inflammatory process caused by various types of pathogens. The aim of the study was to evaluate the local profile of cytokines expressed by native heart valves obtained from patients with acquired heart defects during cardiac surgery. Materials and Methods: As a material for the study, biopsies of native mitral heart valves were obtained from 4 patients with infective endocarditis and from 10 patients with rheumatic heart disease. RNA was isolated using a commercial RNeasy® Plus Universal Mini Kit (Qiagen, Germany), protein extraction was performed using T-PER™ Tissue Protein Extraction Reagent lysis buffer (ThermoScientific, USA) with the addition of protease inhibitors Halt™ Protease Inhibitor Cocktail (ThermoScientific, USA) and 0.5M EDTA solution (ThermoScientific, USA). The level of cytokines expressed by native valves was determined by dot blotting using the Proteome Profiler™ Human Cytokine Array Kit (ARY005B) (R&D Systems, USA). Cytokine gene expression was assessed by qPCR using SYBR Green probes (JSC Evrogen, Russia) on a ViiA7 Real-Time PCR System amplifier (Applied Biosystems, USA). Statistical analysis of the results obtained was performed using the GraphPad Prism 8 program (GraphPad Software, USA). Results: The proteins MIF (macrophage migration inhibitory factor), PAI-1 (plasminogen activator inhibitor-1), ICAM-1 (intercellular adhesion molecule-1) and CXCL12 (stromal growth factor-1) were found to be registered in native valves, obtained from both patients with IE and patients with RHD. Semi-quantitative analysis showed that native valves obtained from patients with IE are characterized by a four-fold increase in the level of PAI-1 secretion and a two-fold decrease in the level of ICAM-1 and CXCL12 compared with the control. MIF was expressed at the same level in both studied groups. IL-1ra (interleukin 1 receptor antagonist), IL-6 (interleukin 6), IL-8 (interleukin 8), IL-16 (interleukin 16), CCL4 (chemokine ligand 4), CCL5 (chemokine ligand 5), and CXCL1 ( chemokine ligand 1) were found only in valves affected by IE. The expression of the MIF, IL6, and IL8 genes was increased in the experimental group relative to the control, and the expression of the PAI1, IL1RA, and CXCL1– genes was decreased in the experiment relative to the control. The gene expression of ICAM1, CXCL12, CCL4, CCL5 and IL16 was the same in both studied groups. Conclusions: The results of the study show that native heart valves affected by IE are characterized by a unique cytokine profile compared to valves obtained from patients with non-infectious endocardial pathology. Native heart valves affected by IE show nonspecific local inflammation, active neovascularization, and decreased resistance to pathogenic bacteremia.

Comparison of different protocols for protein extraction from murine ovarian tissue: A preliminary study

Objective: Extraction of proteins from tissues is a key step to study the protein structure and function. There is no set of universal protocols because of the enormous chemical and physical variety of both proteins and the sample sources. Hence, in this study a comparison of different methods for protein extraction from mouse ovarian tissue was carried out. Methods: Intravaginal dose of 20µl of Phosphate buffer saline was given to 7 mice for ten consecutive days followed by extraction of ovaries. For comparison, seven different protein extraction protocols from Protocol 1 to Protocol 7 were used. The supernatant was first quantified with Bradford assay to determine the total protein yield followed by SDS-PAGE analysis. Results: Highest protein yield of 1.13 mg/ml was attained after supplementing 1% v/v protease inhibitor cocktail in PBS. In total, 13 protein bands were detected by the extraction protocol 2 and 11 protein bands by the extraction protocol 1. 11 protein bands were detected in the range 180 kDa to 22 kDa following Protocol 3 and 4. Extraction with lysis buffer 1 resulted in 7 visible protein bands of molecular weight 182 kDa to 35 kDa range. Only 6 protein bands were observed in the range 182 kDa - 35 kDa following lysis buffer 2 method. For lysis buffer 3, ten visible protein bands were detected in range of 195 kDa - 13 kDa. Conclusion: Based on the protein extraction efficiency of Protocol 1, optimal combination of Phosphate buffer saline (pH 7.2) and protease inhibitor cocktail followed by hand-homogenization is suggested for protein extraction from mouse tissues.

Moisturizing at a molecular level - The basis of Corneocare.

This review covers the last 20 years of research we and our collaborators have conducted on ethnic differences in facial skin moisturization placed in historical context with previous research. We have focussed particularly on the biochemical and cellular gradients of the stratum corneum (SC) with the aim of discovering new skin moisturization and SC maturation mechanisms, identifying new technologies and/or providing conceptual innovations for ingredients that will improve our understanding and treatment of dry skin. Specifically, we discuss gradients for corneodesmosomes and proteases, corneocyte phenotype-inducing enzymes, filaggrin and natural moisturizing factor (NMF), and barrier lipids. These gradients are interdependent and influence greatly corneocyte maturation. The interrelationship between corneodesmolysis and the covalent attachment of ω-hydroxy ceramides and ω-hydroxy fatty acids to the corneocyte protein envelope forming the corneocyte lipid envelope is especially relevant in our new understanding of mechanisms leading to dry skin. This process is initiated by a linoleoyl-ω-acyl ceramide transforming enzyme cascade including 12R lipoxygenase (12R-LOX), epidermal lipoxygenase-3 (eLOX3), epoxide hydrolase 3 (EPHX3), short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7), ceramidase and transglutaminase 1. Our research has opened the opportunity of using novel treatment systems for dry skin based on lipids, humectants, niacinamide and inhibitors of the plasminogen system. It is clear that skin moisturization is a more complex mechanism than simple skin hydration.

A17 EVIDENCE OF PROTEASE-MEDIATED PRO-NOCICEPTIVE EFFECTS OF FECAL SUPERNATANTS FROM CROHN'S DISEASE PATIENTS

Abstract Background Abdominal pain is a debilitating symptom of Crohn’s disease (CD). Despite the current treatment options for this disease, abdominal pain is an unresolved problem that commonly persists in the absence of active inflammation. This suggests that something other than inflammation is driving the pain during the quiescent phase. We have previously reported that microbial proteases can directly modulate the excitability of dorsal root ganglia (DRG) neurons, many of which are pain-sensing. We hypothesize that luminal proteases of CD patients are contributing to their abdominal pain. Purpose Determine whether luminal mediators in CD fecal samples induce changes in pain signalling. Method The effects of patient (active CD [n = 3] and healthy volunteer (HV) [n = 3]) fecal supernatant (FS) samples on pain-sensing neurons were assessed using ex-vivo single unit afferent nerve recordings from mouse colons. Each sample was tested in colonic preparations from a least 5 mice. To further examine cellular mechanisms, DRG neurons were isolated and incubated overnight in media containing CD FS or HV FS media. Changes in neuronal excitability were recorded by determining the rheobase (lower rheobase=increased excitability) using patch clamp recordings (n ≥ 9 DRG neurons/group). Protease inhibitors were applied in both bioassays to determine whether these inhibited the excitatory effect of FS. Lastly, total proteolytic activity in the CD and HV fecal samples was calculated using a casein colorimetric protease detection assay. Result(s) FS from HV had no effect on afferent nerve excitability (p = 0.8920). FS from active CD patients increased action potential discharge from colonic afferent nerves by 85% (p<0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p=0.0074). A protease inhibitor cocktail (1:1000) and protease-activated receptor (PAR)-2 antagonist GB83 (10µM) both blocked the excitatory effects of CD FS (p<0.05). Overnight incubation with CD FS also had an excitatory effect on DRG neurons compared to HV FS (rheobase decreased by 46%, p<0.05). The effect of CD FS was blocked by GB83 (10µM) (p<0.001) and a serine protease inhibitor (FUT175; 100µM) (p<0.05) independently, but the activity was not blocked by E64 (30nM) a cysteine protease inhibitor. A 200-fold increase (p<0.0001) in total proteolytic activity was found in CD FS compared to HV FS. Conclusion(s) Luminal serine proteases, but not cysteine proteases, appear to be driving nociceptive signalling in CD patients. This provides insight into the generation of pain in CD patients and may be a potential target to mitigate this action. Further research is required to elucidate whether these pro-nociceptive proteases are of bacterial or host origin and their effects in the quiescent phase. Disclosure of Interest None Declared

Formulating preservatives to enhance stability of crude extracts of food allergens used for food allergy management and immunotherapy

<abstract> <p>Due to rapid degradation of food allergenic extracts, devising some optimal conditions is mandatory to boost shelf life of extracts for appropriate diagnosis of allergy and immunotherapy. In the current study, food extracts of <italic>Bos domesticus</italic> (cow) milk, <italic>Gallus domesticus</italic> (chicken) egg, <italic>Trititcum aestivum</italic> (wheat), <italic>Gallus domesticus</italic> (chicken) meat and <italic>Arachis hypogaea</italic> (peanut) were prepared and their protein estimation was evaluated by bicinchoninic acid (BCA) method. Stability of food extracts was evaluated under two groups of preservatives; storage with coca's solution (mixture of sodium chloride (NaCl) and sodium bicarbonate (NaHCO<sub>3</sub>)) and stabilizing buffer (sucrose and ethylene diamine tetra-acetic acid (EDTA)). Effect of cocktail of protease inhibitors ((phenyl methyl sulfonyl fluoride (PMSF) and dithiothreitol (DTT)) and glycerol was checked on food extracts under both groups of preservatives. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) was performed for food protein extracts after 1, 3 and 6 months under both conditions. Size of obtained protein bands were compared with allergen database (<ext-link ext-link-type="uri" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.allergen.org">http://www.allergen.org</ext-link>) in order to check their potential allergenicity. Extracts stored with coca's solution remained more potent as compared to stabilizing buffer for up to 3 months except <italic>Gallus domesticus</italic> egg, whose proteins remained more potent in stabilizing buffer as compared to coca's solution. Most of the proteins deteriorated at 6 months of storage. Glycerol had shown better results under both conditions. A formulation containing a combination of coca's solution along with cocktail of protease inhibitors and glycerol improved the shelf life of food extracts for up to 3 months, showing better potential for stabilization of allergenic food extracts and their use for immunotherapy.</p> </abstract>

The effects of cell culture conditions on premature hydrolysis of traceless ester-linked disulfide linkers

Disulfide acids are important for traceless release mechanisms in prodrugs and drug delivery applications. Their ability to self-immolate and release cargo due to environmental stimulus is invaluable. However, complex reactivity patterns may be overlooked as assays increase in complexity or are conducted in media of increasing biological relevance. Conclusions drawn from preliminary characterization in simple phosphate buffers are often applied to in vitro studies in which more complex media are used (e.g. containing glucose, amino acids, FBS, and the cell surface). We developed a model disulfide incorporating a fluorogenic dye as a reporter group in order to explore the generality of the disulfide prodrug system, and used this to explore the stability of disulfide esters in various contexts of increasing complexity. We found that our reporter molecules prematurely released cargo in a series of cell-containing and cell-free assays. We systematically reverse-engineered the components of a complex cell medium and found that FBS was capable of interfering with disulfide-based prodrug linkers, triggering the release of conjugated 4-methylumbelliferone (4-MU) from representative reporter molecules. FBS consistently induced 4-MU release in complete media (i.e. DMEM and RPMI 1640), minimal essential media, and pure water. Signs of 4-MU release were mitigated when FBS was subjected to intense heat (>100 °C) or esterase-specific protease inhibitor cocktail (PIC), indicating that esterases from the serum were capable of triggering cargo release using a hydrolysis mechanism that is separate from the desired reductive cleavage pathway. These findings are important because they show that variance in models may hide unexpected results, which calls for more meticulous consideration of control experiments when developing stimulus-release agents for biological applications.

A Novel Tandem-Tag Purification Strategy for Challenging Disordered Proteins

Intrinsically disordered proteins (IDPs) lack well-defined 3D structures and can only be described as ensembles of different conformations. This high degree of flexibility allows them to interact promiscuously and makes them capable of fulfilling unique and versatile regulatory roles in cellular processes. These functional benefits make IDPs widespread in nature, existing in every living organism from bacteria and fungi to plants and animals. Due to their open and exposed structural state, IDPs are much more prone to proteolytic degradation than their globular counterparts. Therefore, the purification of recombinant IDPs requires extra care and caution, such as maintaining low temperature throughout the purification, the use of protease inhibitor cocktails and fast workflow. Even so, in the case of long IDP targets, the appearance of truncated by-products often seems unavoidable. The separation of these unwanted proteins can be very challenging due to their similarity to the parent target protein. Here, we describe a tandem-tag purification method that offers a remedy to this problem. It contains only common affinity-chromatography steps (HisTrap and Heparin) to ensure low cost, easy access and scaling-up for possible industrial use. The effectiveness of the method is demonstrated with four examples, Tau-441 and two of its fragments and the transactivation domain (AF1) of androgen receptor.

Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: the effects of proteases on laccase activity.

Laccases are enzymes produced by plants and white rot fungi, such as Pleurotus ostreatus, with industrial applications. Fungal laccases have been widely studied, and investigations, such as those involving recombinant DNA technology or adding inducers, have been made to increase laccase production. On the other hand, it has been proposed that extracellular proteases could decrease laccase activity when both types of enzymes are produced by P. ostreatus. The aim of this work was to evaluate the effects of proteases on the activity of extracellular laccases produced by P. ostreatus PoB in submerged culture. Results showed that P. ostreatus PoB produced alkaline, acidic, and neutral proteases. Protease activity was quantified, and the highest activity at alkaline pH (9.0) was 5.63 IU/L (192 h), that at acidic pH (2.0) was 3.38 IU/L (192 h), and that at neutral pH (7.0) was 6.20 IU/L (312 h). The protease activity decreased in the presence of different protease inhibitors, as phenylmethylsulfonyl fluoride (PMSF), EDTA, pepstatin A, and a cocktail of protease inhibitors. Laccase activity was determined in cultures with and without protease inhibitors. In the control culture (without inhibitor), the highest laccase specific activity was 99.88 IU/mg protein. In cultures with PMSF, pepstatin A, or a cocktail of protease inhibitors, laccase activity increased by approximately 1.35-fold (138 IU/mg protein) with respect to the control culture. The inhibitor EDTA did not produce a positive effect on extracellular laccase activity. These results suggest that laccase activity is affected by the actions of acidic and neutral extracellular proteases.

Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry.

Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease.

Analysis of the Proteomes of Primary, De Novo Acute Myeloid Leukemia Cells from Adults

Introduction:Proteins, despite being the primary effectors of cellular processes, are often studied only indirectly through analysis of the transcriptome. However, it is clear that the relationship between mRNA expression and protein expression is approximate at best. In Acute Myeloid Leukemia (AML), the genome and transcriptome have been thoroughly characterized, but the proteome has been less well studied. Here, we present a deep-scale study of the proteomes of 44 primary AML bone marrow samples representing a wide range of AML across the spectrum of cytogenetic risk, common mutations, and driver fusions.Methods:Bone marrow samples were collected at presentation from 44 adult patients with de novo AML as part of an institutional banking protocol, and buffy coat cells were immediately cryopreserved without further manipulation. Cryovials were thawed in the presence of the cell permeable serine protease inhibitor diisopropyl fluorophosphate (DFP) to inactivate the abundant neutrophil serine proteases (ELANE, CTSG, PRTN3, and PRSS57), and further processed for nano-liquid chromatography mass spectrometry in the presence of an extensive cocktail of protease inhibitors. Both label-free quantification (LFQ) and tandem-mass-tag (TMT) deep-scale proteomics were performed on these 44 patient samples, as well as 3 lineage-depleted bone marrow samples from healthy adult donors. Matching RNA-seq and exome sequencing data were available for the same samples as part of The Cancer Genome Atlas (TCGA) AML project.Results:10,651 and 6,679 unique proteins were detected in the TMT and LFQ experiments, respectively. Correlations between measurements derived from the independent proteomic platforms (i.e. TMT and LFQ) is higher (mean Spearman correlation, 0.60, Figure 1A) than correlation between proteomic (TMT) and transcriptomic measurements from bulk RNA-seq data (Spearman 0.43, Figure 1B). Quality checks of the proteomic data strongly supported the reliability of quantification of protein measurements; for example, the mean ratio of beta globin protein (HBB) to alpha globin (HBA1) was 1.2 +/- 0.25 (Figure 1C), and several proteins known to be dysregulated by specific AML-initiating fusion proteins (for PML-RARA, HGF and RARA; for RUNX1-RUNX1T1, RUNX1T1; and for CBFB-MYH11, MYH11) were detected in the expected samples (Figure 1D). Globally, 1,364 proteins were differentially expressed in the AML samples (corrected p-value <0.05, fold change ≥ 1.5) compared to the lineage-depleted, healthy bone marrow samples. Globally overexpressed proteins were enriched for ribosomal RNA modification, mitochondrial protein import, nuclear export, and the mitochondrial electron transport chain, among others. These overexpressed proteins include 61 cell surface proteins that could potentially represent therapeutic targets (overexpressed on average in 82% of AML samples, range 25-97%). Globally downregulated proteins in AML samples were enriched for glycogen metabolism and protein groups associated with mature neutrophils (reflecting the expected maturation block in AML), among others. 771 of the 1364 differentially expressed proteins (56.5%) showed only minimal variability in mRNA expression levels (fold change of <1.1 between AML and normal marrow CD34 cell mRNA) that could not explain dysregulated protein expression. Several protein complexes likewise showed coordinated differential expression in the proteomic data, but no change in the transcriptome, including the THO complex (Figure 1E) and the phosphorylase kinase complex (Figure 1F), among others, indicating the presence of posttranscriptional regulation of the levels of many proteins in AML samples.Conclusion:We have created a deep-scale proteomic database from a set of well-characterized AML samples, allowing for a proteogenomic study of AML. We have identified many examples of post-transcriptional regulation of key metabolic pathways that may be relevant for better understanding AML cell metabolism and therapeutic vulnerabilities. Additional studies linking patterns of protein dysregulation with a variety of AML covariates are underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using protease inhibitors to improve protein stability in the presence of skin: A case study on the stability of insulin like growth factor 1

Insulin-like growth factor 1 (IGF-1) is indicated for growth failure in pediatric patients with primary IGF-1 deficiency and for patients with neutralizing antibodies to growth hormone. IGF-1 was cloned, expressed and purified in-house. Preliminary stability studies prior to the transdermal delivery experiments showed that although stable in contact with stratum corneum, the solution concentration of IGF-1 decreased to 23.63 ± 2.48 and 21.58 ± 2.62% of the initial value upon exposure for 8 h to porcine dermis of 250 and 750 µm thickness. This led to an investigation into how it might be possible to improve the stability of IGF-1 in the presence of porcine/human skin. The stability of IGF-1 in the presence of dermis improved upon heating the skin samples at 60 °C for 2 min suggesting that IGF-1 was subject to enzymatic degradation. Although addition of the protease inhibitor, phenylmethanesulfonyl fluoride (PMSF) alone, did not improve stability, the use of a protease inhibitor cocktail completely blocked proteolytic degradation of IGF-1; the solution concentration after an 8 h exposure to porcine skin was equivalent to the initial level (103.87 ± 9.15%). The results obtained with porcine skin were confirmed with human skin (IGF-1 recovery was 99.31 ± 9.98%). These findings suggest that the inclusion of protease inhibitor cocktails may be useful in limiting the degradation of therapeutic proteins during iontophoresis and transdermal delivery in general – this could be of particular interest for local delivery of peptide/protein therapeutics for dermatological applications.

Addition of Phenylmethylsulfonyl Fluoride Increases the Working Lifetime of the Trout Liver S9 Substrate Depletion Assay, Resulting in Improved Detection of Low Intrinsic Clearance Rates.

The activity of a trout liver S9 substrate depletion assay has been shown to decline over time, presumably due to proteolytic degradation of biotransformation enzymes. To address this problem, assay performance was evaluated following the addition of phenylmethylsulfonyl fluoride (PMSF) or a general-purpose protease inhibitor cocktail to liver homogenization buffers and/or S9 reaction mixtures. Addition of PMSF to liver homogenization buffers and/or S9 reaction mixtures had little or no effect on clearance of phenanthrene, a model cytochrome P450 substrate, in short-term (25 or 30 min) depletion experiments but resulted in significant improvements in retention of this initial activity over time. The protease inhibitor cocktail strongly inhibited initial activity when added to homogenization buffers or reaction mixtures. Taking into consideration potential effects on liver carboxylesterases, the treatment approach determined to be optimal was addition of 10 µM PMSF to the S9 reaction mixture. Addition of 10 µM PMSF to the mixture resulted in significantly higher rates of phenanthrene clearance in 2-h incubations relative to those obtained in the absence of PMSF and a 6-fold increase in the working lifetime of the preparation. The results of a statistical power analysis suggest that by increasing the working lifetime of the assay, addition of PMSF to the reaction mixture could result in substantially improved detection of low in vitro clearance rates when compared to current practice. These findings demonstrate the value of adding PMSF to the trout S9 preparation and may have broad implications for use of this assay to support chemical bioaccumulation assessments for fish. Environ Toxicol Chem 2021;40:148-161. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Time-dependent aggregation of casein micelle concentrates

This research focused on understanding physical and chemical changes occurring to concentrated milk protein suspensions as a function of time. Skim milk (untreated and heat treated at 90°C for 10 min) was concentrated at 6 times the original volume using osmotic stressing, a noninvasive concentration method, maintaining the serum composition as close as possible to that of native milk. A protease inhibitor cocktail, with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases, was added in selected samples. Within 9 d of storage at 4°C, the apparent viscosity increased markedly for both unheated and heated concentrated milk, but not for those in the presence of protease inhibitors. However, only unheated milk showed a significant increase in the apparent diameter of the casein micelles. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry measurements indicated a significantly lower extent of proteolysis in heated than in unheated samples. The microstructure of the aggregates was observed using field emission scanning electron microscopy, and unheated samples clearly showed aggregation of casein micelles with storage time. In heated samples, aggregation was instead triggered by heat-induced protein-protein interactions.

Metabolic changes following transcatheter bariatric embolotherapy for weight loss in obesity: secondary outcomes from a prospective RCT

Abstract Background/Introduction Obesity and its comorbid conditions (i.e. type II diabetes mellitus, atrial fibrillation, coronary artery disease, hypertension, etc...) is a growing burden globally, however, the current treatments (i.e. bariatric surgery, intragasrtic balloons and/or pharmaceutical therapy) pose substantial risks or are contraindicated for various populations. Transcatheter bariatric embolotherapy of left gastric artery by reducing “hunger” hormones from the gastric fundus is a procedure for weight loss that has been growing in prominence over the last several years, however, to date no randomized-controlled trial has been conducted until our study. We studied TBE in a double-blind, sham procedure, first in human RCT of patients (pts) with obesity. Purpose The purpose of this study was to assess the safety and efficacy of TBE for weight loss in obese patients as well as to evaluate metabolic changes. Methods After IV propofol, eligible pts (age 21–60; BMI 35–50 kg/m2) were randomized 1:1 to Sham (skin nick &amp; 1 hr wait) or TBE. All pts received Lifestyle Therapy (behavioral and diet education). Study staff following the pts were also blinded to treatment. Blood samples for gastrointestinal hormones were collected in EDTA tubes containing a protease inhibitor cocktail and frozen per local laboratory standards. All collected samples were assessed together in two batches at the end of the study. The hormones analyzed included ghrelin, GIP, GLP-1, Visfatin, resistin, PAI-1 (total), Leptin, and C-Peptide. An Oral Glucose Tolerance Test (OGTT) and a diabetes assay was performed at baseline and at 6- and 12-months post-intervention. Note, while diabetes was an exclusion criterion for this study, pre-diabetes was not. Results 44 pts were enrolled, of which 40 pts were randomized equally to the groups, with no major complications in either group. TBE demonstrated superior weight loss over the control group at 6- and 12-months post-intervention in both intention-to-treat and per-protocol analyses. At 6 and 12 months, the TBE group demonstrated a clinically meaningful decrease in glucose 1-hour post-fasting by OGTT. GIP levels in the TBE group increased at a mean of 21%, indicative of an improvement in pre-diabetic milieu. Circulating plasma visfatin levels decreased 20% at 6 months and 26% at 12 months in the TBE group indicating a decrease in body fat. C-Peptide levels were noticeably increased in the TBE group at 6 months possibly indicating improvements in insulin sensitivity and beta-cell function. Conclusion(s) TBE is safe and results in clinically significant weight loss and demonstrated a positive effect on glucose homeostasis in pre-diabetic patients. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Endobar Solutions, LLC