Mass Spectrometry Protein Identification Research Articles

Extracellular vesicle response following focused ultrasound-mediated blood–brain barrier opening for the detection of Alzheimer’s pathology

Focused ultrasound (FUS), in conjunction with systemically administered microbubbles, has been shown to induce transient and non-invasive blood–brain barrier opening (BBBO). Originally developed as a method of improving targeted drug delivery, FUS-BBBO has recently been proposed as a means of amplifying detection of disease biomarkers in the bloodstream. We hereby describe a method that can detect extracellular vesicles (EVs) after BBB opening in Alzheimer’s disease (AD) mice and patients. BBBO is shown to enhance the release of EVs with disease-specific cargo to the bloodstream when targeting amyloid affected regions and shown to be more sensitive than cell free DNA. In this presentation, we will describe the overall methodology and quantify the concentration and content of EVs isolated from the serum of mice following drug-free FUS-BBBO in both mice and patients. In AD mice and patients, an average EV concentration increase of 164% and 100% 1 h after FUS-BBBO containing both amyloid and tau was, respectively, found compared to 0% under sham conditions and highly correlated with BBBO volume. Whole genome RNA sequencing and mass spectrometry protein identification demonstrated inflammatory and proliferation gene upregulation. These findings highlight the potential of FUS-BBBO to increase EV serum concentration towards early AD detection.

Shotgun proteomics to characterize wastewater proteins

Classically, the characterization of wastewater components has been restricted to the measurement of indirect parameters (chemical and biological oxygen demand, total nitrogen) and small molecules of interest in epidemiology or for environmental control. Despite the fact that metaproteomics has provided important knowledge about the microbial communities in these waters, practically nothing is known about other non-microbial proteins transported in the wastewater. The method described here has allowed us to perform a large-scale characterization of the wastewater proteome. Wastewater protein profiles have shown to be very different in different collection sites probably reflecting their human population and industrial activities. We believe that wastewater proteomics is opening the doors to the discovery of new environmental and health biomarkers and the development of new, more effective monitoring devices for issues like monitorization of population health, pest control, or control of industry discharges. The method developed is relatively simple and combines procedures for the separation of the soluble and particulate fractions of wastewater and their concentration, and conventional shotgun proteomics using high-resolution mass spectrometry for protein identification.•Unprecedented method for wastewater proteome characterization.•Proteins as new potential biomarkers for sewage chemical-information mining, wastewater epidemiology and environmental monitoring.•Wastewater protein profiles reflect human and industrial activities.

Ammonium sulfate-based prefractionation improved proteome coverage and detection of carbonylated proteins in Arabidopsis thaliana leaf extract

Ammonium sulfate is well known to salt out proteins at high concentrations. The study revealed that it can serve to increase by 60% the total number of identified carbonylated proteins by LC-MS/MS. Protein carbonylation is a significant post-translational modification associated with reactive oxygen species signaling in animal and plant cells. However, the detection of carbonylated proteins involved in signaling is still challenging, as they only represent a small subset of the proteome in the absence of stress. In this study, we investigated the hypothesis that a prefractionation step with ammonium sulphate will improve the detection of the carbonylated proteins in a plant extract. For this, we extracted total protein from the Arabidopsis thaliana leaves and subjected the extract to stepwise precipitation with ammonium sulfate to 40%, 60%, and 80% saturation. The protein fractions were then analyzed by liquid chromatography-tandem mass spectrometry for protein identification. We found that all the proteins identified in the non-fractionated samples were also found in the prefractionated samples, indicating no loss was incurred during the prefractionation. About 45% more proteins were identified in the fractionated samples compared to the non-fractionated total crude extract. When the prefractionation steps were combined with the enrichment of carbonylated proteins labeled with a fluorescent hydrazide probe, several carbonylated proteins, which were unseen in the non-fractionated samples, became visible in the prefractionated samples. Consistently, the prefractionation method allowed to identify 63% more carbonylated proteins by mass spectrometry compared to the number of carbonylated proteins identified from the total crude extract without prefractionation. These results indicated that the ammonium sulfate-based proteome prefractionation can be used to improve proteome coverage and identification of carbonylated proteins from a complex proteome sample.

Proteomic features of skeletal muscle adaptation to resistance exercise training as a function of age.

Resistance exercise training (RET) can counteract negative features of muscle ageing but older age associates with reduced adaptive capacity to RET. Altered muscle protein networks likely contribute to ageing RET adaptation; therefore, associated proteome-wide responses warrant exploration. We employed quantitative sarcoplasmic proteomics to compare age-related proteome and phosphoproteome responses to RET. Thigh muscle biopsies were collected from eight young (25 ± 1.1years) and eight older (67.5 ± 2.6years) adults before and after 20weeks supervised RET. Muscle sarcoplasmic fractions were pooled for each condition and analysed using Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labelling, tandem mass spectrometry and network-based hub protein identification. Older adults displayed impaired RET-induced adaptations in whole-body lean mass, body fat percentage and thigh lean mass (P > 0.05). iTRAQ identified 73 differentially expressed proteins with age and/or RET. Despite possible proteomic stochasticity, RET improved ageing profiles for mitochondrial function and glucose metabolism (top hub; PYK (pyruvate kinase)) but failed to correct altered ageing expression of cytoskeletal proteins (top hub; YWHAZ (14-3-3 protein zeta/delta)). These ageing RET proteomic profiles were generally unchanged or oppositely regulated post-RET in younger muscle. Similarly, RET corrected expression of 10 phosphoproteins altered in ageing, but these responses were again different vs. younger adults. Older muscle is characterised by RET-induced metabolic protein profiles that, whilst not present in younger muscle, improve untrained age-related proteomic deficits. Combined with impaired cytoskeletal adhesion responses, these results provide a proteomic framework for understanding and optimising ageing muscle RET adaptation.

Purification of cytosolic ribosome in Arabidopsis thaliana using different proteases

The ribosome is a macromolecular system that has a significant role in synthesizing protein within all living cells. Ribosome in eucaryotic cell consists of two subunits; including small (40S) and large (60S) ribosomal subunits. Moreover, each subunit contains one or more molecules of ribosomal RNA (rRNA) and several ribosomal proteins (r-protein). The purification and isolation of ribosomal proteins from the other cellular organelles are a highly complicated process which needs to do several purification steps to get the desired ribosomal proteins. In this study, the cytosolic ribosomal proteins in the Arabidopsis thaliana cell culture have been purified from other organelles. Also, several types of proteases have been studied to find the best one that hydrolyzes peptide bonds that are used in mass spectrometry protein identification applications. The results have shown that by using double addition of sucrose cushion into the purified sample and using trypsin enzyme, we could purify large number of ribosomal proteins in the sample.

Abstract 40: SARS-CoV-2/ACE2 Induces Vascular Inflammatory Responses In Human Microvascular Endothelial Cells Independently Of Viral Replication

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), p<0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4±18 vs. C: 1011±268 RFU, p<0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFκB activation. Proteomics analysis of the global effect of rS1, identified biological process enrichment of proteins from virus transcription and NFκB signalling. ACE2 Co-IP identified 216 interacting proteins (filtered with ≥1 unique peptide, 1% FDR), linked to cytokine production and inflammation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. In conclusion, we demonstrate that rS1p induces an inflammatory response through ACE2 in endothelial cells. These effects seem to be independent of viral infection. Our findings suggest that vascular inflammation in COVID-19 involves activation of ACE2-mediated pro-inflammatory signalling that may be unrelated to viral replication.

Lacritin bactericidal peptide N‐104 targets FeoB and PotH through interaction with the surface‐exposed lipoprotein YaiW

Putrescine and iron transporters PotH and FeoB play roles in bactericidal mechanism of tear lacritin peptide ‘N-104’. Given that N-104 doesn't appear to disrupt bacterial lipid membranes, we asked how N-104 bactericidal activity involves the inner-membrane proteins in Gram-negative bacteria, for example, ocular pathogenic P. aeruginosa PA14. We synthesized N-104, and negative control lacritin peptide C-95 with cysteine added to the N-terminus, and coupled them to SulfoLink® Coupling Resin. Overnight cultures of PA14 were washed in PBS, lysed and incubated overnight (4ºC) in lysis buffer containing 200 mM octyl β-D-glucopyranoside (‘OG’) to allow membrane proteins getting into OG vesicles. The total lysate supernatant was passed through PierceTM Agarose Resin precolumns prior to overnight incubation (4ºC) with N-104 or C-95 columns. After washing nonspecifically bound proteins, interacting proteins were eluted by gradient addition of 50, 75, 100, 125, 150, 300, 500 or 1000 mM KCl. The 500 mM KCl eluted proteins from the two columns were subjected to mass spectrometry for protein identification. The surface-exposed lipoprotein ‘YaiW’ was the sole identified outer-membrane protein hit with 7.4x higher enrichment from N-104 vs negative control C-95 columns, while no hits were obtained from biotinylated surface proteins of PA14 (possibly due to amine-reactive interference). We know that YaiW contributes to the transport of proline-rich Bac7 peptides across the outer membrane, thus YaiW appears to be a receptor for N-104 and potentially capable of transporting the N-104 into the periplasm. We also studied bactericidal efficacy of N-104 against PA14 in the free living nematode C. elegans, as a model mammalian system. Adult worms grown on a lawn of E. coli strain OP50 were harvested in M9 buffer. Approximately, 107 cfu/ml of bacteria strains, including PA14, PA14 feoB mutant, PA14 potH mutant (from PA14NR Set; Harvard), and OP50 as a negative control were untreated or treated with 10 and 20 μM N-104 in 0.1xPBS for 5 hours (35°C). 75-100 worms were incubated with the cells in sterile black 96-well plate with the final composition of 90% M9 buffer and 10% LB broth (in a humidified chamber at room temperature). After 3 days, bacteria and debris were aspirated from the wells and worms were stained with 2.5 μM SYTOX Orange in M9 and imaged next day by capturing both transmitted (all worms) and epi-fluorescence (dead worms) images. While all PA14 strains (wild-type, feoB mutant, and potH mutant), but not OP50, were able to kill >75% of the worm population, N-104 treated samples of wild-type PA14 couldn't damage any worms. In contrast, N-104 treated samples of feoB mutant, and potH mutant strains could kill about 35% and 25% of the worm populations confirming the roles for PotH and FeoB in N-104's bactericidal mechanism.

Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n=2) and Cryptosporidium hominis (n=1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.

Pancreatic Ductal Adenocarcinoma Arising in Young and Old Patients Displays Similar Molecular Features.

Simple SummaryPancreatic ducal adenocarcinoma (PDAC) is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years old (y.o.). Multiple molecular defects such as DNA damages and oxidative stress present in PDAC are associated with ageing. With a multiomics approach, we assessed the molecular features of early-onset tumors (≤55 y.o.) and compared them to classical late-onset tumors (≥70 y.o.). Our results demonstrated that tumors from both groups showed a similar molecular profile. Given that young patients are more often included in clinical trials, this absence of difference is an important finding that supports younger patients as a good molecular surrogate model for the general older population of patients with PDAC.Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years (y.o.). While the genomic and transcriptomic landscapes of late-onset tumors (LOT) have been described, little is known about early-onset tumors (EOT). Ageing is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT (≤55 y.o.) and 316 LOT (≥70 y.o.) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defenses together with increased proteome carbonylation. However, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbor a comparable molecular profile to that of LOT.

The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis.

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains’ phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains’ phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.

PDAC Arising in Young and Old Patients Display Similar Molecular Features

Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade. Yet, approximately 10% of patients are diagnosed under 55 years. While the genomic and transcriptomic landscapes of late onset tumors (LOT) have been described, little is known about early onset tumors (EOT). Aging is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT ( 70 years) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defences together with increased proteome carbonylation. Yet, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbour a very comparable molecular profile to that of LOT. Funding Statement: This research was funded by Nelia and Amadeo Barletta Foundation. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The hospital ethics committee approved this study (IRB 00003835-2010/01NCIB).

Isolation and characterisation of Leishmania donovani protein antigens from urine of visceral leishmaniasis patients.

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.

Phage G Structure at 6.1 Å Resolution, Condensed DNA, and Host Identity Revision to a Lysinibacillus

Phage G has the largest capsid and genome of any known propagated phage. Many aspects of its structure, assembly, and replication have not been elucidated. Herein, we present the dsDNA-packed and empty phage G capsid at 6.1 and 9 Å resolution, respectively, using cryo-EM for structure determination and mass spectrometry for protein identification. The major capsid protein, gp27, is identified and found to share the HK97-fold universally conserved in all previously solved dsDNA phages. Trimers of the decoration protein, gp26, sit on the 3-fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G's decoration protein is longer than what has been reported in other phages, and we suspect the extra interaction surface area helps stabilize the capsid. We identified several additional capsid proteins, including a candidate for the prohead protease responsible for processing gp27. Furthermore, cryo-EM reveals a range of partially full, condensed DNA densities that appear to have no contact with capsid shell. Three analyses confirm that the phage G host is a Lysinibacillus, and not Bacillus megaterium: identity of host proteins in our mass spectrometry analyses, genome sequence of the phage G host, and host range of phage G.

Isobaric Labeling Strategy Utilizing 4-Plex N,N-Dimethyl Leucine (DiLeu) Tags Reveals Proteomic Changes Induced by Chemotherapy in Cerebrospinal Fluid of Children with B-Cell Acute Lymphoblastic Leukemia.

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

Identification of soybean trans-factors associated with plastid RNA editing sites.

RNA editing is a posttranscriptional process that changes nucleotide sequences, among which cytosine-to-uracil by a deamination reaction can revert non-neutral codon mutations. Pentatricopeptide repeat (PPR) proteins comprise a family of RNA-binding proteins, with members acting as editing trans-factors that recognize specific RNA cis-elements and perform the deamination reaction. PPR proteins are classified into P and PLS subfamilies. In this work, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform trans-factor specific protein isolation. Soybean cis-elements from these three different RNA probes show differences in respect to other species. Pulldown samples were submitted to mass spectrometry for protein identification. Among detected proteins, five corresponded to PPR proteins. More than one PPR protein, with distinct functional domains, was pulled down with each one of the RNA probes. Comparison of the soybean PPR proteins to Arabidopsis allowed identification of the closest homologous. Differential gene expression analysis demonstrated that the PPR locus Glyma.02G174500 doubled its expression under salt stress, which correlates with the increase of its potential rps14 editing. The present study represents the first identification of RNA editing trans-factors in soybean. Data also indicated that potential multiple trans-factors should interact with RNA cis-elements to perform the RNA editing.

Proteomic Discovery: Elevated Neurogranin Levels in Children with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited hemoglobinopathy that frequently causes neurological complications, such as stroke, silent cerebral infarct (SCI) and other forms of brain injury, including loss of cognition. SCI is defined as any ischemic lesion visible on T2-weighted magnetic resonance imaging (MRI) that is not associated with a focal neurologic deficit in the same vascular distribution, and is the most commonly recognized form of brain injury in SCD. SCI is associated with decreased neurocognitive function and increased risk for new or enlarging SCI/stroke. The purpose of this study was to use mass spectrometry (MS) based proteomics to discover and validate plasma brain proteins associated with brain injury in children with SCD. The plasma samples used for the proteomics discovery analyses were from two groups: screening samples of participants in the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) (n=15), and 6 age-matched, healthy non-SCD controls, three of whom had sickle cell trait. Samples from children with SCD were divided into two groups: those with SCI (n=7) and those without SCI (n=8) matched for age, hemoglobin (Hb) and white blood cell counts. To identify circulating markers of SCI, plasma proteomes were analyzed using a sequential separation approach of Hb and top abundant plasma protein depletion, followed by reverse phase separation of intact proteins, trypsin digestion and tandem MS. Neurogranin (NRGN), a small calmodulin-binding synaptic protein (PMID 9886843), was the most abundant brain-enriched protein found in the plasma of children with SCD. We developed a NRGN sandwich immunoassay for verification of MS analysis results in an independent cohort from the SIT Trial longitudinal samples. For this verification study, plasma samples from the SIT Trial and 25 normal controls were tested for NRGN levels at study entry and exit by immunoassay. The majority of the SCD samples were obtained from an ancillary study to collect longitudinal samples that started after the SIT Trial had begun. There was no overlap between the discovery and the verification group samples. The normal control samples were from 25 healthy, age and gender comparable non-SCD pediatric controls unrelated to the SIT Trial, without evidence of acute/chronic illness. From the discovery SCD group, 1172 unambiguous proteins were identified; 639 proteins were identified from normal controls, as shown in Figure 1. Twenty-five percent (289/1172) of these proteins were found solely in the SCI group. Our MS protein identification data were filtered against this list of expressed brain proteins to produce a composite list of brain proteins found in plasma from children with SCD, but not found in plasma from age, gender and race-matched healthy control children. Twenty-five plasma proteins known to be expressed in one or more cell type enriched in the human brain based on bioinformatics searches were more abundant in the SCD vs control groups, as shown in Table 1. Median NRGN levels were higher at study entry in children with SCD (0.44 ng/mL, N=98), in comparison to a healthy, non-SCD control group (0.12 ng/mL, N=25, p < 0.0001), as shown in Figure 2. Using the longitudinal samples from the SIT Trial, there were no differences in NRGN levels between the observation group and the treatment group (p=0.35) over time. There was no significant difference in median NRGN levels between the observation group and the treatment group at the initial visit (0.36 vs 0.19, p=0.069) or final visit (0.39 vs 0.52, p=0.52). Using the initial visit samples from the SIT Trial, there was no significant difference in median NRGN levels between the non-SCI (n=35) and SCI groups (n=61) (p=0.073). There was no significant correlation between NRGN levels and neuropsychological measures of executive function (rho=0.089, p=0.58) and IQ (rho=-0.081, p=0.62). Our non-biased proteomic discovery studies demonstrate that NRGN levels are increased in children with SCD, when compared to normal children. Additional studies will be necessary to determine whether NRGN levels correlate with specific forms of neurologic injury. Further validation studies of larger cohorts with a multi-analyte ELISA of these brain proteins identified in plasma (Table 1) are warranted, and could help establish possible links between brain-specific proteins and neurocognitive outcomes. Disclosures Lance: NIH: Research Funding; KKI: Research Funding. Faulcon:FDA: Employment, Other: Although the author is a FDA/CTP employee, this work was not done as part his/her official duties. This publication reflects the views of the author and should not be construed to reflect the FDA/CTP's views or policies.. Fu:GSK: Employment. Yang:ImmunArray: Patents & Royalties: ImmunArray. Strouse:Global Blood Therapeutics: Consultancy. Barron-Casella:Mast Pharmaceudicals - spouse: Consultancy; ImmunArray - spouse: Patents & Royalties. Van Eck:ImmunArray: Consultancy, Patents & Royalties. Casella:NIH: Research Funding; Mast Pharmaceuticals: Consultancy; Immunoarray Ltd.: Patents & Royalties. Everett:ImmunArray: Consultancy, Patents & Royalties.

Phosphoproteins Involved in the Inhibition of Apoptosis and in Cell Survival in the Leiomyoma.

Uterine leiomyomas are benign smooth muscle cell tumors originating from the myometrium. In this study we focus on leiomyoma and normal myometrium phosphoproteome, to identify differentially phosphorylated proteins involved in tumorigenic signaling pathways, and in anti-apoptotic processes and cell survival. We obtained paired tissue samples of seven leiomyomas and adjacent myometria and analyzed the phosphoproteome by two-dimensional gel electrophoresis (2-DE) combined with immobilized metal affinity chromatography (IMAC) and Pro-Q Diamond phosphoprotein gel stain. We used mass spectrometry for protein identification and Western blotting for 2-DE data validation. Quantities of 33 proteins enriched by the IMAC approach were significantly different in the leiomyoma if compared to the myometrium. Bioinformatic analysis revealed ten tumorigenic signaling pathways and four phosphoproteins involved in both the inhibition of apoptosis and cell survival. Our study highlights the involvement of the phosphoproteome in leiomyoma growth. Further studies are needed to understand the role of phosphorylation in leiomyoma. Our data shed light on mechanisms that still need to be ascertained, but could open the path to a new class of drugs that not only can block the growth, but could also lead to a significant reduction in tumor size.

Characterization of the molecular changes associated with the overexpression of a novel epithelial cadherin splice variant mRNA in a breast cancer model using proteomics and bioinformatics approaches: identification of changes in cell metabolism and an increased expression of lactate dehydrogenase B

BackgroundBreast cancer (BC) is the most common female cancer and the leading cause of cancer death in women worldwide. Alterations in epithelial cadherin (E-cadherin) expression and functions are associated to BC, but the underlying molecular mechanisms have not been fully elucidated. We have previously reported a novel human E-cadherin splice variant (E-cadherin variant) mRNA. Stable transfectants in MCF-7 human BC cells (MCF7Ecadvar) depicted fibroblast-like cell morphology, E-cadherin wild-type downregulation, and other molecular changes characteristic of the epithelial-to-mesenchymal transition process, reduced cell-cell adhesion, and increased cell migration and invasion. In this study, a two-dimensional differential gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS) protein identification and bioinformatics analyses were done to characterize biological processes and canonical pathways affected by E-cadherin variant expression.ResultsBy 2D-DIGE and MS analysis, 50 proteins were found differentially expressed (≥ Δ1.5) in MCF7Ecadvar compared to control cells. Validation of transcript expression was done in the ten most overexpressed and underexpressed proteins. Bioinformatics analyses revealed that 39 of the 50 proteins identified had been previously associated to BC. Moreover, metabolic processes were the most affected, and glycolysis the canonical pathway most altered. The lactate dehydrogenase B (LDHB) was the highest overexpressed protein, and transcript levels were higher in MCF7Ecadvar than in control cells. In agreement with these findings, MCF7Ecadvar conditioned media had lower glucose and higher lactate levels than control cells. MCF7Ecadvar cell treatment with 5 mM of the glycolytic inhibitor 2-deoxy-glucose led to decreased cell viability, and modulation of LDHB expression in MCF7Ecadvar cells with a specific small interfering RNA resulted in decreased cell proliferation. Finally, a positive association between expression levels of the E-cadherin variant and LDHB transcripts was demonstrated in 21 human breast tumor tissues, and breast tumor samples with higher Ki67 expression showed higher LDHB mRNA levels.ConclusionsResults from this investigation contributed to further characterize molecular changes associated to the novel E-cadherin splice variant expression in BC cells. They also revealed an association between expression of the novel variant and changes related to BC progression and aggressiveness, in particular those associated to cell metabolism.

An Evaluation of Palladin Overexpression in Pancreatic Cancer Cells

Pancreatic cancer (PC) is the fourth leading cause of cancer in United States with overall survival rates of less than 5% (ACS, 2018). Its aggressive nature and late onset of physical symptoms lead to poor prognoses. Recognition of biomarkers is clearly an imperative undertaking for the disease. To tackle this problem, we evaluated the secreted protein profile of pancreatic cancer cell lines derived from different stages. In total, nine human pancreatic cancer cell lines were successfully adapted into serum‐free environments from which conditioned media was collected by a combination of ultracentrifugation, molecular weight cutoff, protein precipitation, gel‐based separation, and tryptic digestion followed by mass spectrometry protein identification. A small subset of proteins has been identified and validated biochemically including Palladin (PALLD), a pancreatic cancer susceptibility protein shown to be up‐regulated in previous studies. PALLD is a scaffolding protein that supports remodeling of the actin cytoskeleton in association with integrins at focal adhesion sites to coordinate cell migration. We demonstrate a possible interaction between PALLD and the beta5 integrin (ITGB5) indicative of the increased motile phenotype in PC. Taken together, our results suggest that PALLD overexpression is associated with enhanced motility in pancreatic cancer cells and could represent a potential biomarker or therapeutic strategy against pancreatic cancer progression.Support or Funding InformationThis work was funded in part through intramural programs at Fresno State to KV, RCB, JAB, and through NCI P20CA138025 to JAB.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two-Dimensional Electrophoresis and Mass Spectrometry for Protein Identification.

Two-dimensional electrophoresis is useful for separation of individual proteins that can be easily isolated and processed with mass spectrometry for their identification. Here we describe a simplified method of 2D electrophoresis with aim to help beginners in optimization of protein separation protocol, which can be further modified to enhance protein spot resolution. Current protocol can be used in different fields of biology including progenitor cells.