Micrograms Of Protein Research Articles

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the “Submerge and Dry” protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

Modular automated bottom-up proteomic sample preparation for high-throughput applications.

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with protein relative quantity variance (CV%) below 15%.

The proteome of distal nerves: implication in delayed repair and poor functional recovery.

Chronic denervation is one of the key factors that affect nerve regeneration. Chronic axotomy deteriorates the distal nerve stump, causes protein changes, and renders the microenvironment less permissive for regeneration. Some of these factors/proteins have been individually studied. To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect, we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model. Four rats that received immediate repair after sciatic nerve transection served as control, whereas four rats in the experimental group (chronic denervation) had their sciatic nerve repaired after a 12-week delay. All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins. Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels. NanoLC-MS/MS analysis was applied to the gels. Protein expression levels of nerves on the surgery side were compared to those on the contralateral side. Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed. All the differentially expressed proteins in both groups were further stratified according to the biological processes. A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration. Ingenuity Pathway Analysis (IPA) software was used for pathway analysis. The results showed that 709 differentially expressed proteins were identified in the delayed repair group, with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified. The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated. IPA revealed that protective pathways such as LXR/RXR, acute phase response, RAC, ERK/MAPK, CNTF, IL-6, and FGF signaling were inhibited in the delayed repair group, whereas three detrimental pathways, including the complement system, PTEN, and apoptosis signaling, were activated. An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types. The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

Adding polyvinylpyrrolidone to low level protein samples significantly improves peptide recovery in FASP digests: An inexpensive and simple modification to the FASP protocol

Filter-aided sample preparation (FASP) remains a popular choice for proteomic sample preparation, particularly for its ability to produce a 'clean' peptide sample clear of large molecule contaminants. However, sample loss continues to be a problem particularly for sample inputs that contain less than ten micrograms of protein. Here, we describe that the simple addition of a polymer, polyvinylpyrrolidone-40 (PVP-40) to the protein sample prior to FASP digest significantly improves peptide recovery and identifications, especially with lower level sample inputs. PVP-FASP produces clean samples which required no additional sample clean-up prior to nanoLC-MS analysis. In addition, PVP-FASP is compatible with other FASP modifications, including the use of sodium deoxycholate (DOC) to improve trypsin digestion. SIGNIFICANCE: Simple modification to FASP procedure improves sample recovery during proteomic digests in SDS, improving peptide identifications and median peptide intensity.

Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The importance of the brain renin angiotensin system (RAS) in blood pressure (BP) regulation and water homeostasis is well accepted. However, it remains unclear whether renin and/or its precursor, prorenin, are expressed in the brain and whether they trigger signaling pathways in specific brain regions. We previously used double transgenic mice expressing enhanced green fluorescent protein controlled by renin promoter and beta‐galactosidase controlled by the human angiotensinogen (AGT) promoter to localize their expression in the brain. We reported that neurons expressing renin are localized in close proximity to AGT‐expressing cells in the rostral ventrolateral medulla (RVLM), a brainstem nucleus involved in sympathetic activity and BP control. Here, we hypothesized that prorenin induces phosphorylation of intracellular cascades such as extracellular signal‐regulated kinases (ERK1/2) and activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) in the brainstem. Neonatal brainstem cells were cultured from C57BL/6J mice at postnatal day 1–3 and maintained for 7–10 days in B27‐supplemented neurobasal medium. Immunofluorescence experiments demonstrated the cultures contain both MAP2+ neurons and GFAP+ astrocytes. Expression of type 1 angiotensin receptor (AT1R), renin, and prorenin receptor (PRR) was confirmed by qPCR. To quantify ERK1/2 phosphorylation, cells were stimulated with recombinant prorenin (rProrenin) at 100 nM for 0, 30, and 60 min. Total and phosphorylated ERK1/2 were detected by Western Blot. The effect of rProrenin on NADPHox activity was measured by lucigenin chemiluminescence assay. Specific activity of NADPHox was expressed as chemiluminescence units per min per microgram of protein. Vehicle and phorbol myristate acetate were used as negative and positive controls, respectively. Incubation with rProrenin increased the ratio of phosphorylated‐to‐total ERK1/2 from 0.65±0.07 A.U. at baseline to 1.01±0.13 A.U. at 30 min (54% increase; p=0.0035; n=7) and 1.29±0.09 A.U. at 60 min (102% increase; p<0.0001; n=7). We generated preliminary data showing that both PRO20, a PRR blocker, and Ro‐31, a protein kinase C inhibitor, abrogated prorenin‐induced ERK1/2 phosphorylation. In an additional experiment, we observed that Angiotensin II (Ang II) at 100 nM induced ERK1/2 phosphorylation from 0.98 A.U. to 1.52 A.U. at 15 min (55 % increase), 1.61 A.U. at 30 min (65% increase), and 1.35 A.U. at 60 min (38% increase). Incubation of cells with 100 nM rProrenin for 6 hours resulted in 47.2±9.5 % induction of NADPHox activity compared to vehicle control (p<0.05, n=5). Finally, fluorescent in situ hybridization (RNAscope) data suggests that PRR is expressed in the RVLM neurons and RVLM‐targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane‐anesthetized mice. We conclude that prorenin in the brainstem induces downstream signaling involving ERK/2 phosphorylation and generation of ROS, which might contribute to BP elevation and sympathoexcitation. Future studies will determine whether prorenin‐elicited signaling in the brainstem requires binding to PRR, generation of Ang II, and/or AT1R.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

Experimental Protein-Containing Preparations <i>Streptococcus pneumoniae</i>, Obtained from Fresh Isolated Strains and Museum

Relevance. Vaccines based on capsular polysaccharides of pneumococci are not active against serotypes that are not included in the vaccine, non-capsulated strains and do not protect against carriage caused by other serotypes. Their use leads to the replacement of dominant serotypes of pneumococci, the appearance of highly virulent strains, changes in the microbial landscape of mucous membranes due to the appearance of other etiologically significant pathogens of respiratory tract diseases. This requires the creation of intraspecific anti-pneumococcal immunity, which will be facilitated by the development of serotype-dependent drugs, which will include protein-containing antigens of pneumococci. Objective. Study of serotype-independent activity of protein-containing antigenic components obtained from freshly isolated and archival strains of S. pneumoniae. Materials and methods. Strains of three serotypes of S. pneumoniae were used: archival strains of serotypes 6B N296, 19F N 298 and 10A N297 and freshly isolated serotypes 6B N 1121, 19F N 1055 and serogroup 10 N 1193 (from the cerebrospinal fluid of patients with purulent meningitis).. In the experimental protein-containing preparations EPCP obtained, the protein content was determined. Protective activity of EPCP and virulence of strains were determined in the model of intraperitoneal immunization and infection of BALB/c mice; LD50 was calculated using the generally accepted modified Kerber formula. The immunophenotype of lymphocytes previously isolated from donor-mice whole blood was studied by flow cytometry. Statistical analysis of the materials was carried out using parametric and nonparametric methods using the application package «Statistica for Windows», ver. 7.0 (Stat Soft, Inc); in statistical analysis, the significance level of p was assumed to be < 0.05. Results and discussion. During cultivation there was the analysis of growth dynamics showed intensive accumulation of biomass of the archival strain N296 with low virulence, and lower - by virulent strain N 1121 isolated from the patient's cerebrospinal fluid. The protein content of drugs from serotype 6B strains did not differ. The strains of other serotypes isolated during the generalized infectious process were also more virulent than the archival strain. The effect on the immunophenotype of human lymphocytes of fractions of 50-100 kDa isolated from the initial preparations obtained by culturing serotype 6B, significantly increased only under the influence of the preparation from the archival strain. In the study of protective activity of the initial preparations from strains of serotype 6B and fractions with MM 50-100 kDa only triple immunization with the initial preparation from strain N 296, at a dose of 20 micrograms of protein per mouse, led to significantly greater survival of immunized mice from infection with the newly isolated virulent strain N 1121 of homologous serotype. A fraction of 30-100 kDa provided protection of mice twice immunized with 50 mkg of protein per mouse, with a high efficacy index of 8.9, even after infection with a freshly isolated strain of heterologous S. pneumoniae serotype 3 N 10196. Conclusion. The protein-containing fraction with MM 30-100 kDa obtained from a low virulent archival strain of S. pneumoniae serotype 6B N296 possessed protective activity against a newly isolated virulent strain of heterologous serotype after double immunization. Under the action of the studied protein-containing fractions, activation of the cellular component of immune system with the involvement of innate immunity effectors and T-lymphocytes is shown. These data can be considered as a evidence for further study of EPCP to assess the possibility of their use in the design of anti-pneumococcal drug with serotype-independent protective activity.

Abstract GS2-05: Microscaled proteogenomic methods for precision oncology

Abstract Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a tumor (>10,000 proteins) and more than >20,000 phosphosites starting with just 25 micrograms of protein per sample. In order to understand the capabilities and limitations our our approach relative to more conventional deepscale proteomics requiring >10X more starting material, we compared preclinical patient derived xenograft (PDX) models at conventional scale with data obtained by core-needle biopsy of the same tissues. Comparable depth and biological insights were obtained from the cores relative to surgically resected tumors. As a proof-of-concept for implementation in clinical trials, we applied microscaled proteogenomic methods to a small-scale clinical study where biopsies were accrued from patients with ERBB2+ locally advanced breast cancer before and 48 to 72 hours after the first dose of neoadjuvant Trastuzumab-based chemotherapy. Multi-omics comparisons were conducted between samples associated with residual disease versus samples associated with complete pathological response. Integrative, microscaled proteogenomic analyses efficiently diagnosed the molecular bases of diverse candidate treatment resistance mechanisms including: 1) absence of ERBB2 amplification (false-ERBB2+); 2) insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2+); 3) resistance features in true-ERBB2+ cases including androgen receptor signaling, mucin expression and an inactive immune microenvironment; 4) lack of acute phospho-ERBB2 down-regulation in non-pCR cases. In summary, we have developed a robust proteogenomics pipeline well suited for large-scale cancer clinical studies to identify potential resistance mechanism in patients. We conclude that microscaled cancer proteogenomics could improve diagnostic precision in the clinical setting. Citation Format: Shankha Satpathy, Eric Jaehnig, Krug Karsten, Beom-Jun Kim, Alexander Saltzman, Doug Chan, Kimberly Holloway, Meenakshi Anurag, Chen Huang, Purba Singh, Ari Gao, Noel Namai, Yongchao Dou, Bo Wen, Suhas Vasaikar, David Mutch, Mark Watson, Cynthia Ma, Foluso Ademuyiwa, Mothaffar Rimawi, Jeremy Hoog, Samuel Jacobs, Anna Malovannaya, Terry Hyslop, D.R Mani, Charles Perou, George Miles, Bing Zhang, Michael Gillette, Steven Carr, Matthew Ellis. Microscaled proteogenomic methods for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-05.

182 Bisphenol A, but not bisphenol S, affects key microRNAs during bovine oocyte maturation

Oocyte maturation involves crucial hormone-dependent events that are uniquely susceptible to toxic insults by endocrine disrupting chemicals (EDCs). Emerging evidence suggests that small RNAs, including microRNAs (miRNAs), may be key participants in the response to EDCs. Bisphenol A (BPA) and bisphenol S (BPS) are chemicals with detrimental health effects, with BPA negatively affecting oocyte quality. The mode of action of bisphenols at the epigenetic level is not clear. Several miRNAs have been identified as crucial regulators of gene expression during development. This study aimed to examine key miRNAs in response to BPA or BPS treatment during oocyte maturation. Primary forms (pri-miRNA) and mature forms of miR-21, miR-155, miR-34c, and miR-146a were quantified by quantitative (q)PCR in IVM bovine cumulus-oocyte complexes (COCs) and invitro cultured (IVC) cumulus cells treated with BPA and BPS at physiologically significant doses (0.05mgmL−1). Total RNA, containing both forms of microRNAs, was isolated from pools of 40 COCs on a minimum of three biological replicates. Primary miRNAs and mature miRNAs were reverse transcribed (RT) using qScript cDNA and microRNA/cDNA kits, respectively, and quantified by qPCR, with three technical replicates for each biological one. In addition, mRNA and protein quantification of the downstream target DNMT3A in IVC cumulus cells further enhanced our understanding of EDC interference in epigenetic regulations in female reproduction. Total RNA was isolated from cumulus cells, mRNA (1μg) and mature miRNAs (0.5μg) were RT separately and cDNA was quantified by qPCR, as described above. Expression values were normalized against two housekeeping genes selected by GeNorm analysis. Twenty micrograms of proteins extracted by sonication were loaded on a 8% acrylamide gel and analysed by western blotting. Densitometry analysis was performed on 3 separate blots with protein levels normalized to the loading control, β-actin. One-way ANOVA was used to determine statistical differences among treatment groups with P<0.05 considered statistically significant. Results showed that BPA significantly increased miR-21 in COCs (P=0.02) and cumulus cells (P=0.01), increased pri-miR-21 in oocytes (P=0.03), suppressed miR-34c in cumulus cells (P=0.02), increased miR-155 in denuded oocytes (P=0.04), and had no effect on miR-146a. No changes were observed in response to BPS. Experiments in IVC cumulus cells showed similar miRNA profiles: miR-21 and miR-155 were significantly overexpressed in BPA-treated cells (P=0.04); however, miR-34c and miR-146a were not affected. Messenger RNA levels of DNMT3A increased (P=0.02) and protein levels of DNMT3A decreased in response to BPA (P=0.005). Overall, this study presents novel findings of BPA-induced miRNA dysregulation in IVM bovine oocytes and in IVC bovine cumulus cells. We can speculate that miR-21 participates in the BPA-induced dysregulation of DNMT3A, contributing to decreased fertility. This study did not show any effect of BPS on microRNA expression, suggesting an alternative mechanistic pathway for this analogue.

P7. Moldable allograft bone gel infused with spine-derived exosomes triggers osteogenic induction

BACKGROUND CONTEXT Exosomes are nanoscale vesicles that function as critical mediators of cell-to-cell communication via transportation of molecular cargo from a source cell to a target cell. It has previously been shown that the differentiation fate of primitive cells, such as mesenchymal stem cells (MSCs), can be modified toward an osteogenic path by the uptake of exosomes from defined cell types. Due to these properties, exosomes derived from a bone source such as spine seem an appropriate tool to guide the osteogenic differentiation of progenitor cells. PURPOSE A moldable bone gel was developed to serve as an osteoconductive support in filling bone voids. The effects of processing bone into a gelatinous material inhibits its innate osteoinductive properties. In an effort to restore the osteoinductive capacity of the bone gel product, spine-derived exosomes were added to the gel composition to restore bone graft material osteoinductivity and enhance handling. STUDY DESIGN/SETTING Purified exosomes were characterized by flow cytometry using coating latex beads with the nanoparticles and subsequently labeling these beads with known exosome markers, CD63, CD81 and CD9. Scanning Electron Microscopy (SEM) was performed to verify the size and morphology of the exosomes. Exosome concentration was determined using EXOCET Exosome Quantitation Kit (System Biosciences). PATIENT SAMPLE Exosomes were obtained from qualified cadaveric human spines by ultrafiltration and subsequent differential ultracentrifugation (Beckman Optima XE-90 Ultracentrifuge equipped with a SW32 rotor) of the clarified supernatant. OUTCOME MEASURES Scanning electron microscopy (SEM) was performed to verify the size and morphology of the exosomes. Exosome concentration was determined using EXOCET Exosome Quantitation Kit (System Biosciences). Protein concentration was determined using a Qubit 4.0 Fluorometer (Thermo Fisher), from which the purity of the exosome sample was determined by calculating exosome concentration per microgram of protein. A sensitive, quantitative method to assess the bone forming potential of C2C12 myoblast cell line was used. The expression of alkaline phosphatase, a widely accepted bone marker, was measured following treatment of C2C12 cells with spine-derived exosomes alone or in combination with bone gel using polycarbonate membrane transwells. METHODS Treatment with 50ng of BMP-4 was used as a positive control. Alkaline phosphatase expression was normalized to total protein content, which was measured with Pierce BCA Protein Assay Kit (Thermo Fisher). The osteoinductive (OI) index was calculated by using the following formula: (OI test sample result – OI negative control result)/OI negative control result/protein concentration. An index over 20% of negative baseline was considered as osteoinductive. RESULTS Spine-derived exosomes positively expressed the exosome flow cytometry markers tested. Specifically, they expressed 99 ± % of CD81, 85 ±14% of CD63 and 64 ± 35% of CD9. SEM imaging revealed most of the exosomes were approximately 100 nm in size, consistent with the expected physiological size range of exosomes (30 – 150nm). The mean concentration of the spine-derived exosomes obtained was 1.22 ± 1.09 × 1010 exosomes/mL of supernatant. The mean number of exosomes per microgram of protein was 3.31 ± 2.33 × 108 indicating relatively high purity. Osteoinductive testing was performed using different concentrations of exosomes either alone or in combination with bone gel. The OI index of treatment of C2C12 cells with BMP-4 or 2 × 108, 1 × 109, 2 × 109, 5 × 109 or 1 × 1010 exosomes alone was 28.5, 1.0, 3.7, 7.4, 11.8 and 27.6 respectively. The OI index of treatment with 2 × 108, 1 × 109, 2 × 109, 5 × 109 or 1 × 1010 exosomes, with each dose combined with 0.25cc of bone gel, was 0.9, 4.5, 6.2, 9.3 and 18.5 respectively. These results revealed a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression. All doses were 20% above negative baseline indicating an osteoinductive effect at doses ranging from 2 × 108 to 1 × 1010 exosomes alone or with bone gel. All data is expressed as mean ± SEM from 3 separate experiments. Statistical analysis was performed using Student's t-test or one-way ANOVA followed by Bonferroni's post hoc test if multiple group comparisons were performed. CONCLUSIONS In this preliminary study, in vitro osteoinductive effect of spine-derived exosomes alone or infused in bone gel on C2C12 cells was demonstrated. Although different concentrations of exosomes were tested, a single concentration (50ng) of BMP-4 was used as a positive control. Future studies will evaluate in vivo activity in a bone defect rat model. There are specific and key miRNA transcripts involved in the observed osteoinductive regulation to better understand the molecular mechanism of exosome-delivered therapy in the context of bone regeneration. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

A Fully Integrated Spintip-Based Approach for Sensitive and Quantitative Profiling of Region-Resolved in Vivo Brain Glycoproteome.

Region- and cell type-resolved global proteome and specific post-translational modifications (PTMs) profiling of tissues has drawn great attention recently for interpreting the heterogeneous multicellular microenvironment of various in vivo systems. Due to access to low microgram of proteins and low abundance of glycoproteins, spatially resolved glycoproteome analysis of in vivo tissue sections remains challenging. Several glycoproteomics sample preparation strategies were established for processing microgram-level of protein samples, but these strategies were not either fully integrated or directly compatible with tissue samples when considering protein extraction in strong lysis buffers. Moreover, these approaches mainly focused on identification of glycosylation sites, but pay less attention to quantification, all of which limit their applications. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieves all the steps of glycoprotein enrichment, digestion, deglycosylation, and desalting in single spintip device. Sample loss is significantly reduced, and the total processing time is reduced to 4 h, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 μg of mouse brain proteins. By seamlessly combining with laser capture microdissection (LCM), the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1875, 1794, 1801, and 1417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of in vivo tissue sections.

Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics.

Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust, and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis. This technique builds upon the single-pot solid-phase-enhanced sample preparation (SP3) where we now demonstrate its scalability (low to high micrograms of protein) and the influence of variables such as bead and enzyme amounts on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extraction resulting in a more reliable method that is simple and easy to implement for routine and high-throughput analysis of proteins. We benchmarked the performance of this technique against filter-aided sample preparation (FASP) using 30 μg of total HeLa protein lysate. We also show that the USP3 method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 μg of HeLa protein lysate. The USP3 protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and analysis by MS.

IL‐15 Upregulates GLUT1 while Reducing CPT1 in Cardiomyocytes

Obesity is an exponentially growing epidemic in the United States and is a leading contributor of cardiovascular disease (CVD). Maintenance of lifestyle interventions to prevent and treat obesity, such as diet and exercise, is a challenge. In this regard, targeting the molecular mediators of obesity has become a focus to prevent CVD disease progression. In an undiseased state, the cardiac muscle relies primarily on oxidative processes for energy production. In some cases, CVD can lead to upregulation of glycogenic, rather than oxidative, processes to produce ATP for muscle contraction. The myokine interleukin‐15 (IL‐15) is secreted by skeletal muscle during exercise and has been shown to reduce obesity and related disorders. However, the effects of IL‐15 signaling in cardiomyocytes (CM) remains largely unknown. The objective of the current study was to determine if IL‐15 regulates glucose and/or fatty acid (FA) metabolism in CM. Differentiated CM were treated with 100 ng/mL of IL‐15 for 24 hours. Protein was harvested, from either IL‐15 or vehicle treated cells, and isolated into total, mitochondrial and plasma membrane (PM) fractions. The protein concentration was quantified utilizing a Bradford assay. Subsequently, protein analysis, via Western blotting, was performed. Fifteen micrograms of protein was loaded onto 4–20% polyacrylamide gels for electrophoresis. The proteins were then transferred onto a PVDF membrane. Primary antibodies against GLUT1, GLUT4, and CPT1 (1:1000) were used, followed by secondary antibodies (1:2000). GLUT1 and GLUT4 were analyzed in both total cell fractions and PM fractions, while CPT1 levels were assessed in mitochondrial fractions. The membranes were subjected to enhanced chemiluminescence, imaged and quantified with ImageJ software. A t‐test (P<0.05) was used to determine if IL‐15 had any significant effects on the protein levels of the aforementioned proteins. IL‐15 did not alter total GLUT4, PM GLUT4 or PM GLUT1 (P>0.05). Conversely, IL‐15 treatment resulted in a 367% increase in total GLUT1 protein levels when compared to the vehicle control cells (P<0.05). Of note, IL‐15 decreased mitochondrial CPT1 by nearly 50% (P<0.05). Our data indicate that IL‐15 increases the protein concentration of one of the glucose transporters, GLUT1, and decreases the protein concentration of the mitochondrial FA transporter, CPT1, in CM. Thus, IL‐15 may be responsible for promoting a shift in CM metabolism from FA to glucose utilization. Additional studies are warranted to confirm this proposed shift in metabolism by studying glucose uptake and beta‐oxidation activity in CM. Further, to understand if an IL‐15‐mediated shift in metabolism is beneficial to CM, further studies aimed at assessing cardiac muscle function are required. Taken together, if IL‐15 holds the potential as a treatment for obesity, examination of its functions in various tissues is required.Support or Funding InformationChapman University Crean College of Health and Behavioral Sciences Summer Undergraduate Research Fellowship; American Heart Association 16SDG30680003This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity

summary In the absence of extrinsic adjuvants, purified Amb a 1 induced a strong Th2-polarized immune response in mice. This high allergenic activity seems to be linked to the allergen’s susceptibility to endolysosomal proteolysis.

3D-SISPROT: A simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology for deep proteome profiling

Multidimensional peptide fractionation strategies have been approved as the efficient approaches to significantly improve the depth of proteome coverage. In this study, a simple and integrated spintip-based protein digestion and three-dimensional peptide fractionation technology (3D-SISPROT) was developed for the deep proteome profiling from low microgram of proteins as starting materials. All the sample preparation steps, including protein digestion, strong anion exchange (SAX)-based fractionation, and high-pH reversed phase (RP) fractionation were integrated into one pipette tip packed with SAX and C18 membranes for the first time. The SAX plus C18 membranes design minimizes the sample loss and ensures high efficient SAX-based digestion. 4275 proteins were identified with 1.4h of MS time when 6μg cell lysates was processed. More importantly, the SAX-based digestion procedure did not influence the SAX-based peptide fractionation efficiency which was done in the same SAX membrane. The 3D-SISPROT was exemplified by the analysis of 30μg of HEK 293T cell lysates with 20.4h of MS time, which resulted in the identification of 8222 proteins including 3215 annotated membrane proteins. Gene Ontology annotations indicated that the 3D-SISPROT was unbiased for the proteins from major cellular components. Taking advantages of the efficient SAX-based and high-pH RP-based fractionation strategies, we expect that the 3D-SISPROT can be applied for the deep proteome profiling with limited starting material.

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles

BackgroundChemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation. MethodsWe utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay. ResultsApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV. ConclusionsApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation. General significanceThis study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.

Trehalose prevents aggregation of exosomes and cryodamage.

Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry. Here we report that addition of 25 mM trehalose to pancreatic beta-cell exosome-like vesicle isolation and storage buffer narrows the particle size distribution and increases the number of individual particles per microgram of protein. Repeated freeze-thaw cycles induce an increase in particle concentration and in the width of the size distribution for exosome-like vesicles stored in PBS, but not in PBS 25 mM trehalose. No signs of lysis or incomplete vesicles were observed by cryo-electron tomography in PBS and trehalose samples. In macrophage immune assays, beta-cell extracellular vesicles in trehalose show consistently higher TNF-alpha cytokine secretion stimulation indexes suggesting improved preservation of biological activity. The addition of trehalose might be an attractive means to standardize experiments in the field of exosome research and downstream applications.

A novel microscale preparative gel electrophoresis system.

Currently, the separation targets of preparative electrophoresis range from milligrams to micrograms of proteins. However, most commercially available preparative electrophoretic instruments function at the milligram level. Although some preparative electrophoretic apparatuses operated at the microgram level, the fractionation results are often unsatisfying because they suffer from low resolution, poor recovery, or a long fractionation time. To address these issues, we developed a novel microscale preparative electrophoresis system that consists of a separation apparatus and an elution apparatus. Protein samples are first loaded onto the separation apparatus and separated over the gel according to the molecular weight of each protein. Then the separation gel is transferred to the elution apparatus and the proteins on the gel are eluted through the thickness of the gel. This system offers the following advantages: (1) high resolution: almost no overlap between the adjacent fractions; (2) a short recovery time: fractionation was performed in 2 hours including separation in 100 min and elution in 20 min and (3) high recovery: recovery was as high as 91.8%.

Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.

Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.