SEQUEST Search Research Articles

COL1A2 Barcoding: Bone Species Identification via Shotgun Proteomics.

Species identification of fragmentary bones remains a challenging task in archeology and forensics. A species identification method for such fragmentary bones that has recently attracted interest is the use of bone collagen proteins. Here, we describe a method similar to DNA barcoding that reads collagen protein sequences in bone and automatically determines the species by performing sequence database searches. The method is almost identical to conventional shotgun proteomics analysis of bone samples, except that the database used by the SEQUEST search engine consisted only of entries for collagen type 1 alpha 2 (COL1A2) proteins from various vertebrates. Accordingly, the COL1A2 peptides that differ in sequence among species act as species marker peptides. In SEQUEST-based shotgun proteomics, the protein entries that contain more marker peptide sequences are assigned higher scores; therefore, the highest-scoring protein entry will be the COL1A2 entry for the species from which the analyzed bone was derived. We tested our method using bone samples from 30 vertebrate species and found that all species were correctly identified. In conclusion, COL1A2 can be used as a bone protein barcode and can be read through shotgun proteomics, allowing for automatic bone species identification. Data are available via ProteomeXchange with the identifier PXD045402.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

Digging deeper into the immunopeptidome: characterization of post-translationally modified peptides presented by MHC I

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8–12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5–7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.

PRiSM: A prototype for exhaustive, restriction-free database searching for mass spectrometry-based identification.

The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification. We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification. Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously. Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics.

Obscurin and Clusterin Elevation in Serum of Acute Myocardial Infarction Patients

We discovered new biomarker candidates for acute myocardial infarction (AMI) using micro LC and accurate mass spectrometry. Two serum samples were pooled and used for biomarker screening. UHPLC‐LTQ‐Orbitrap analysis was used with data‐dependent acquisition followed by Sequest search. Six biomarker candidates were selected after fold change analysis of peptides in AMI patient samples compared to healthy controls. Literature search results were reflected. The six biomarker candidates were characterized and quantified using multi reaction monitoring using synthesized peptides or trypsin digested protein standards. Levels of obscurin, alpha‐1‐acid glycoprotein 1, and clusterin were significantly increased (p < 0.001) in AMI patient samples compared to levels in healthy controls, while those of antithrombin III and fibrinogen beta were not. We confirmed the diagnostic performance of obscurin and clusterin as biomarker candidates for AMI using commercial ELISA kits. Receiver operating characteristic results showed that obscurin and clusterin showed highest sensitivity and specificity for AMI diagnosis together with troponins by both of multi‐reaction monitoring analysis and ELISA.

Identification of Host-Response in Cerebral Malaria Patients Using Quantitative Proteomic Analysis.

The objective of this study was to study the altered proteome in the frontal lobe of patients with CM. Unbiased analysis of differentially abundant proteins could lead to identification of host responses against Plasmodium falciparum infection, which will aid in better understanding of the molecular mechanism of pathophysiology in CM. TMT-based quantitative proteomic analysis using high-resolution mass spectrometry is employed. In brief, proteins are isolated from frontal lobe samples, which are collected at autopsy from three cases of CM and three control subjects. Equal amounts of protein from each case are digested using trypsin and labeled with different TMT reagents. The pooled sample is fractionated using strong cation exchange chromatography and analyzed on Orbitrap Fusion in triplicates. For accurate quantitation of peptides, the samples are analyzed in MS3 mode. The data is searched against a combined database of human and P. falciparum proteins using Sequest and Mascot search engines. A total of 4174 proteins are identified, of which, 107 are found to be differentially abundant in the test samples with significant p-value (<0.05). Proteins associated with biological processes such as innate immune response, complement system, coagulation, and platelet activation are found to be elevated in CM cases. In contrast, proteins associated with myelination, oxidative phosphorylation, regulation of reactive oxygen species, and sodium and calcium ions transport are found to be depleted in response to CM. In addition, three P. falciparum proteins exclusively in CM brain samples are also identified. The study signifies neuronal assault due to axonal injury, altered sodium and calcium ion channels, deregulated inflammation and demyelination as a part of host response to CM. Enhanced oxidative stress, repressed oxidative phosphorylation, and demyelination of axons may contribute to the severity of the disease. Further validation of these results on a large cohort can provide leads in the development of neuroprotective therapies for CM.

Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry

α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and tryptic peptides T2 (3CFITPDITSK12) and T4 (24TWCDAFCSIR33) were monitored for detection and confirmation of CTX. The limit of detection (LOD) was 0.05ng/mL for CTX in plasma, and the limit of confirmation (LOC) 0.2ng/mL. Unlike small peptides consisting of the 20 canonical amino acids, CTX was stable in equine plasma at ambient temperature for at least 24h. The developed analytical method was successfully applied to analysis of incurred plasma samples; CTX was detected in plasma collected 15min through 36h post subcutaneous administration of CTX (2.0mg dose) to a research horse, and confirmed 30min through 24h. Additionally, an approach named “reliable targeted SEQUEST search” has been proposed for assessing the specificity of T2 at product ion spectrum level for confirmation of CTX. T2 is uniquely specific for CTX, as evaluated with this approach and BLAST search. Furthermore, the effect of dimethyl sulfoxide (DMSO) as a mobile phase additive on electrospray (ESI) response of T2 and T4, background noise level and signal to noise ratio (S/N) was examined; DMSO increased signal intensity of T2 and T4 by a factor of less than 2. It is the first report that DMSO raised background noise level and did not improve S/N for the peptides, to the authors’ knowledge. The developed analytical method may be applicable for analysis of CTX in plasma from other species such as greyhound dogs or even human beings.

Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells.

4-amino-2-trifluoromethyl-phenyl retinate (ATPR) was reported to potentially inhibit proliferation and induce differentiation activity in some tumor cells. In this study, a proteomics approach was used to investigate the possible mechanism by screening the differentially expressed protein profiles of SGC-7901 cells before and after ATPR-treatment in vitro. Peptides digested from the total cellular proteins were analyzed by reverse phase LC-MS/MS followed by a label-free quantification analysis. The SEQUEST search engine was used to identify proteins and bioinformatics resources were used to investigate the involved pathways for the differentially expressed proteins. Thirteen down-regulated proteins were identified in the ATPR-treated group. Bioinformatics analysis showed that the effects of ATPR on 14-3-3ε might potentially involve the PI3K-AKT-FOXO pathway and P27Kip1 expression. Western blot and RT-PCR analysis showed that ATPR could inhibit AKT phosphorylation, up-regulate the expression of FOXO1A and P27Kip1 at both the protein and mRNA levels, and down-regulate the cytoplasmic expression of cyclin E and CDK2. ATPR-induced G0/G1 phase arrest and differentiation can be ablated if the P27kip1 gene is silenced with sequence-specific siRNA or in 14-3-3ε overexpression of SGC-7901 cells. ATPR might cause cell cycle arrest and differentiation in SGC-7901 cells by simultaneously inhibiting the phosphorylation of AKT and down-regulating 14-3-3ε. This change would then enhance the inhibition of cyclin E/CDK2 by up-regulating FOXO1A and P27Kip1. Our findings could be of value for finding new drug targets and for developing more effective differentiation inducer.

Confirmatory analysis of etanercept in equine plasma by LC-MS for doping control.

Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmune-based diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by anti-human IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then digested by trypsin. Tryptic peptides (T1 from human tumour necrosis factor receptor 2 of etanercept, T15 and T27 from human IgG1 of the protein) were employed for detection and confirmation of the analyte. The limit of detection was 5 ng/mL, and the limit of confirmation 10 ng/mL. This method is specific for confirmation of etanercept, as assessed using the results from BLAST and SEQUEST searches. The results from SEQUEST searches also revealed an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for identification of etanercept. It is the first report for such a finding, to the authors' knowledge. The method was successful in analyses of the plasma samples collected post administration of etanercept to horses. Etanercept was detected up to 11 days post administration. This method will be helpful for confirmation of etanercept or other protein-based drugs consisting of human IgG1, in equine drug testing. Copyright © 2016 John Wiley & Sons, Ltd.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

Proteomic Analysis of the Excretory and Secretory Proteins of Haemonchus contortus (HcESP) Binding to Goat PBMCs In Vivo Revealed Stage-Specific Binding Profiles.

Haemonchus contortus is a parasitic gastrointestinal nematode, and its excretory and secretory products (HcESPs) interact extensively with the host cells. In this study, we report the interaction of proteins from HcESPs at different developmental stages to goat peripheral blood mononuclear cells (PBMCs) in vivo using liquid chromatography-tandem mass spectrometry. A total of 407 HcESPs that interacted with goat PBMCs at different time points were identified from a H. contortus protein database using SEQUEST searches. The L4 and L5 stages of H. contortus represented a higher proportion of the identified proteins compared with the early and late adult stages. Both stage-specific interacting proteins and proteins that were common to multiple stages were identified. Forty-seven interacting proteins were shared among all stages. The gene ontology (GO) distributions of the identified goat PBMC-interacting proteins were nearly identical among all developmental stages, with high representation of binding and catalytic activity. Cellular, metabolic and single-organism processes were also annotated as major biological processes, but interestingly, more proteins were annotated as localization processes at the L5 stage than at the L4 and adult stages. Based on the clustering of homologous proteins, we improved the functional annotations of un-annotated proteins identified at different developmental stages. Some unnamed H. contortus ATP-binding cassette proteins, including ADP-ribosylation factor and P-glycoprotein-9, were identified by STRING protein clustering analysis.

Dataset of UV induced changes in nuclear proteome obtained by GeLC-Orbitrap/MS in Pinus radiata needles.

Although responses to UV stress have been characterised at system and cellular levels, the dynamics of the nuclear proteome triggered in this situation are still unknown, despite its essential role in regulating gene expression and in last term plant physiology. To fill this gap, we characterised the variations in the nuclear proteome after 2 h and 16 h (8 h/day) of UV irradiation by using state-of-the-art mass spectrometry-based shotgun proteomics methods combined with novel bioinformatics workflows that were employed in the manuscript entitled “The variations in the nuclear proteome reveal new transcription factors and mechanisms involved in UV stress response in Pinus radiata” (Pascual et al., 2016) [1]. We employed in-gel digestion followed by a 120 min gradient prior to MS analysis. Data was processed following two approaches: a database dependent employing the SEQUEST algorithm and custom databases, and a database independent by mass accuracy precursor alignment (MAPA). 388 proteins were identified by SEQUEST search and 9094 m/z were quantified by MAPA. Significant m/z were de novo sequenced using the Novor algorithm. We present here the complete datasets and the analysis workflow.

Abstract 1179: Unique metabolic profile of Vemurafenib-resistant melanoma cells: a quantitative proteomics approach

Abstract Malignant melanoma is noted for its aggressive clinical behavior and propensity for lethal metastasis. Despite the remarkable success of BRAF inhibitor Vemurafenib in clinics, emergence of resistance remains a limiting factor in increasing overall survival. Thus, comprehensive studies are needed to increase the biological understanding of BRAF-resistant tumors in order to develop effective therapeutic regimens. To study the molecular mechanism(s) involved in acquired resistance, we generated a Vemurafenib-resistant cell line in the BRAFV600E mutated SK-Mel28 melanoma cell background. Incubation of SK-Mel28 cells with Vemurafenib (0.1 μM) resulted in an initial loss of cell viability after which majority of cells resumed proliferation. Thereafter, cells were exposed to gradually increasing Vemurafenib concentrations and allowed to proliferate in the presence of 5 μM Vemurafenib, to generate the A2-1b resistant cell line. Viability assays established the IC50 of parental SK-Mel28 cells as 0.2 μM, while IC50 &amp;gt; than 12 μM was noted for the Vemurafenib-resistant A2-1b population. We utilized a quantitative proteomics strategy, employing label-free nano-LC-MS/MS technology on a Q-exactive, to compare the proteome of A2-1b with SK-Mel28 cells. The data searched against the human proteome using the Sequest search engine and further analyzed with SIEVE software resulted in the positive identification of 1720 proteins (≤1% FDR). This data set containing proteins with only uniquely identified peptides with a high level of confidence (p&amp;lt;0.05) was uploaded into Ingenuity Pathway Analysis and Panther softwares, with number of peptides and corresponding ratio between the two groups, set as observational parameters. Notably, the largest fraction (49%) of identified proteins belonged to cellular metabolic processes. Glycolysis, gluconeogenesis and NADH repair were the major pathways modulated in Vemurafenib-resistant A2-1b cells. In addition, TCA cycle and unfolded protein response was significantly affected. A subset of 13 proteins, linked to these pathways and not previously associated with acquired resistance was identified. The upregulated proteins included CALD1, OR4A15, PTPRK, CALU, IDH3A, RNH1, CLIC4, SPAG17, SND1 and VCP while ENO2, TPI1 and GAPDH were significantly downregulated. In summary, the proteomic approach applied for the first time to study acquired resistance to BRAF inhibition identified novel targets which upon further validation in appropriate models has the potential to provide valuable therapeutic strategies against Vemurafenib-resistant melanomas. Citation Format: Deeba N. Syed, Rahul K. Lall, Iram Majeed, Feng Liu, Frank L. Meyskens, Hasan Mukhtar. Unique metabolic profile of Vemurafenib-resistant melanoma cells: a quantitative proteomics approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1179. doi:10.1158/1538-7445.AM2015-1179

A draft map of rhesus monkey tissue proteome for biomedical research.

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.

Differential protein expression in compatible and incompatible pollen-pistil interactions in almond [Prunus dulcis (Miller) D. A. Webb] by 2D-DIGE and HPLC-MS/MS

SummarySelf-incompatibility (SI) in almond [Prunus dulcis (Miller) D.A.Webb] is of the gametophytic-type and is controlled by the S-locus, which is highly polymorphic and includes two tightly-linked genes expressed in the pistil (as an SRNase) and in the pollen [as an S haplotype-specific F-box (SFB) protein]. Experimental evidence indicates that other proteins are also needed for the functioning of self-incompatibility. However, to date, S-RNase and SFB are the only components of the SI system identified in almond. Moreover, the cause(s) of a lack of S-RNase activity in selfcompatible almonds remain(s) unclear. This work aimed to identify the other proteins involved in the self- (in)compatible response in almond by comparative quantitative analysis of the differential expression of proteins following incompatible and compatible pollinations. Four self-incompatible almond cultivars were self-pollinated or cross-pollinated, and a homozygous self-compatible almond selection was self-pollinated. Proteins were extracted from the pollinated pistils and analysed by two-dimensional difference gel electrophoresis (2D-DIGE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Searches were performed against the NCBI database using the MASCOT and SEQUEST search engines and Proteome Discoverer v1.4 software. A total of 43 proteins, some with a key role in pollen-pistil interactions, were identified as being differentially expressed following incompatible or compatible pollination. These results provide the first profiles of differential protein expression during self-(in)compatibility in almond.

Altered Cellular Responses As Biomarkers for Fluoranthene Exposure to the Bone Marrow-Mesenchymal Stem Cells: Implications for Stem Cell-Based in Vitro Models That Help Determine the Associated Hematotoxicity

Background: Fluoranthene is a member of the polycyclic aromatic hydrocarbon family, comprising ubiquitous environmental pollutants and potent mutagens. Fluoranthene has been classified by the International Agency for Research on Cancer as a group 3 carcinogens, whose carcinogenicity has not been fully determined in humans. Moreover, the stem cell toxicity and global hematotoxicity associated with fluoranthene exposure have not been thoroughly studied in the bone marrow-mesenchymal stem cells (BM-MSCs). In this study, we determined whether fluoranthene-induced cellular responses could be used as biomarkers for the examination of BM-MSC dysfunction, and suggested the possible use of BM-MSCs for monitoring the acute hematotoxicity caused by environmental hazards.Materials and Methods: Apreviously published protocol was used for the isolation and characterization of BM-MSCs. Fluoranthene was added to the cell culture medium in the range of 25–500 µM. The cellular levels of hydrogen peroxide, indicating the presence of reactive oxygen species, were measured using an enzyme immunoassay. The mitochondrial mass, membrane potential, and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, MitoTracker Red probes, and real time PCR, respectively. A proteomic analysis of the mitochondrial-rich cytoplasmic fraction was performed using nano-LC-ESI-MS/MS, BioWorksBrowser, and the SEQUEST search engines. Quantitative mRNA and immunoblot measurements were used to further confirm the altered mRNA expression as well as to determine the levels of cellular proteins obtained from the proteomic analysis.Results: After exposure to fluoranthene, the BM-MSCs showed a marked reduction in cell number, and the viability decreased substantially after two days of exposure. BM-MSCs that were not treated with fluoranthene remained compact and spindle-shaped. These cells remained tightly attached to each other and to the substrate. In general, a direct exposure of fluoranthene depressed the proliferative capacity and altered the cell morphology of BM-MSCs. The cells detached from the subsurface, and cell-to-cell attachments were also lost. The viability significantly decreased after two days of fluoranthene exposure. The mtDNA copy number and the mass showed a rapid elevation after a 5-day exposure to fluoranthene. Hundreds of cellular proteins in the mitochondria-rich cytoplasmic fraction were markedly deregulated in cells treated with fluoranthene. The protein expression levels of poly [ADP-ribose] polymerase 1 (PARP-1), elongation factor 1-gamma, heat shock 70 kDa protein 1A/1B, heterogeneous nuclear ribonucleoproteins A2/B1 isoform B1, ATP-dependent RNA helicase DDX5, and T-complex protein 1 subunit theta were upregulated more than five-fold in cells treated with fluoranthene than in untreated cells. A significant (more than 2-fold) down-regulation in the cellular levels of the proteins myosin-9, protein ALO17 isoform 1, filamin-C isoform b, Na/K-transporting ATPase subunit alpha-1, nuclear pore membrane glycoprotein 210, and DNA-dependent protein kinase catalytic subunit isoform 2 was observed after a similar fluoranthene treatment. The presence of PARP-1 was further confirmed using mRNA analysis.Conclusion: This study investigated the global cellular responses after exposure to fluoranthene: PARP-1 was recognized as a notable biomarker for monitoring the PAH-induced hematotoxicity. In summary, BM-MSCs are promising candidates for the development of unique in vitro model systems for predicting fluoranthene-associated hematotoxicity and general toxicity in humans.Keywords: Fluoranthene, genotoxicity, hematotoxicity, bone marrow-mesenchymal stem cells [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Profiling Of Polycyclic Aromatic Hydrocarbons-Induced Genotoxicity In Peripheral Blood Stem Cells and Bone Marrow Mesenchymal Stem Cells

BackgroundPolycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that are potent mutagens and classified as carcinogens by International Agency for Research on Cancer. However, genotoxic effect of PAHs exposure has not been thoroughly presented in peripheral blood stem cells (PBSC) and bone marrow-mesenchymal stem cells (BM-MSC). Therefore, this study investigated the profiling of PAHs-induced genotoxicity in hematopoietic and mesenchymal stem cells using primary human-derived PBSC and BM-MSC. Zebrafish (Danio rerio) as in vivo vertebrate model was used for monitoring genotoxicity of PAHs exposure to the PBSC and BM-MSC. Materials and MethodsCytokine stimulation (10 mg/kg/d) was used in three healthy male donors from 5 d before to 2 d after. One to three apheresis procedures were planned for on day 0, day 1, and day 2, using a Baxter CS-3000 PLUS machine (Baxter Healthcare) and a COBE-Spectra (Gambro) for obtaining PBSC. Previous published protocol was used for the isolation and characterization of BM-MSCs. For in vitro cell line study, cells were cultured and maintained in RPMI media containing 10% fetal bovine serum and four types of PAHs- benzopyrene (BaP), pentacene, fluoranthene and pyrene - were added in the cell culture media with 100µM concentration. Cellular level of hydrogen peroxide as a representative marker of reactive oxygen species was measured using enzyme immunoassay. Mitochondrial mass, membrane potential and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, Red probes and real time PCR, respectively. Proteomics in mitochondrial-rich cytoplasmic fraction was performed using nano-LC-ESI-MS/MS analysis with the BioWorksBrowser (Thermo Fisher Scientific Inc., CA) and the SEQUEST search engines. For in vivo model study, Zebrafish embryos 30h post-fertilization (hpf) were exposed to BaP at concentrations of 200, 400, 600, 800 and 1000nM. Seventy embryos were cultured in 40 mL BaP solution in each petri dish, and there were three replicates for each of the five treatments. Embryos were collected at 54 hpf, 78hpf and 102hpf for the study of genotoxicity and morphological changes. ResultsAccording to each type of PAHs, the results of cell biology study showed different aspects: fluoranthene displayed profound significant reduction in cell count, especially in PBSC and viability decreased substantially after two days of exposuring into fluoranthene. On the third day of PAHs exposure, viability reduced remarkably in all the cells. Each type of cells displayed different proportionality of apoptosis. The proportion of apoptosis and cellular ROS level were increased in time- and dose-dependent manner after 3 days of PAHs treatment with different degree levels according to the type of PAHs. Mitochondrial contents and membrane potentials were increased with different pattern: mtDNA copy number and mass were dramatically elevated after 5-day treatment of fluoranthene and pyrene in both stem cells and in vivo Zebrafish model. Several hundreds of cellular proteins in mitochondrial-rich cytoplasmic fraction were profoundly deregulated in comparison to control group. The notable deregulated proteins for PAHs exposure were displayed as following: prelamin-A/C isoform 3 and annexin A1 for benzopyrene; prelamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly [ADP-ribose] polymerase 1 (PARP-1) for fluoranthene; talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, prelamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities in the general shape of Zebrafish, including curved backbone and cardiomegaly, were observed in the 54 hpf with more than 400 nM of BaP. ConclusionThis study showed global profiling of toxic changes of PAHs in PBSC and BM-MSC as well as Zebrafish model. The change of mitochondrial genome (increased mtDNA copy number and mass) was closely associated with PAH exposure in hematopoietic and mesenchymal stem cells. Among cellular proteins, prelamin-A/C isoform 3, talin-1 and annexin A1 were remarkably elevated after exposure of PAHs, this may play a role as biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs. Disclosures:No relevant conflicts of interest to declare.

Large precursor tolerance database search — A simple approach for estimation of the amount of spectra with precursor mass shifts in proteomic data

Mass measurement and precursor mass assignment are independent processes in proteomic data acquisition. Due to misassignments to C-13 peak, or for other reasons, extensive precursor mass shifts (i.e., deviations of the measured from calculated precursor neutral masses) in LC-MS/MS data obtained with the high-accuracy LTQ-Orbitrap mass spectrometers have been reported in previous studies. Although computational methods for post-acquisition reassignment to monoisotopic mass have been developed to curate the MS/MS spectra prior to database search, a simpler method for estimating the fraction of spectra with precursor mass shift so as to determine whether the data require curation remains desirable. Here, we provide the evidence that an easy approach, which applies a large precursor tolerance (2.1Da or higher) in SEQUEST search against a forward and decoy protein sequence database and then filters the data with PeptideProphet peptide identification probability (p≥0.9), could detect most of the MS/MS spectra containing inaccurate precursor masses. Furthermore, through the implementation of artificial mass shifts on 4000 randomly selected MS/MS spectra, which originally had accurate precursor mass assigned by the mass spectrometers, we demonstrated that the accuracy of the precursor mass has almost negligible influence on the efficacy and fidelity of peptide identification. Integral precursor mass shift is a known problem and thus proteomic data should be handled and analyzed properly to avoid losing important protein identification and/or quantification information. A quick and easy approach for estimating the number of MS/MS spectra with inaccurate precursor mass assignments would be helpful for evaluating the performance of the instrument, determining whether the data requires curation prior to database search or should be searched with specific search parameter(s). Here we demonstrated most of the MS/MS spectra with inaccurate mass assignments (integral or non-integral changes) that could be easily identified by database search with large precursor tolerance windows.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Identification of urinary biomarkers for type 2 diabetes using bead-based proteomic approach

AimsTo seek urinary peptides as biomarkers distinguishing type 2 diabetes mellitus (T2DM) patients from healthy controls. MethodsRandom urine samples obtained from 28 patients with T2DM and 29 healthy individuals were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) after purification using weak cationic-exchange magnetic beads (MB-WCX). Then the generated mass spectra of peptides were analyzed by ClinProTools2.1 bioinformatics software. Subsequently, the amino acid sequences of differently expressed peptides were identified by a nano-liquid chromatography–tandem mass spectrometry and a Sequest search found the corresponding protein name. ResultsThree differently expressed peptides and their mass to charge ratios (m/z) were found. Compared with healthy controls, the peak areas of the three differently expressed peptides were all reduced in T2DM, and the m/z were 1056.1 (m/z), 1963.5 (m/z), 2123.5 (m/z), respectively. The above-mentioned peptides were further identified as fragments of histidine triad nucleotide-binding protein 1 (HINT1), bifunctional aminoacyl-tRNA synthetase (EPRS), and clusterin precursor protein (CLU). ConclusionsHistidine triad nucleotide-binding protein 1, bifunctional aminoacyl-tRNA synthetase, and clusterin precursor protein may serve as potential biomarkers distinguishing type 2 diabetes mellitus patients from healthy controls.