Proteome Of Peripheral Blood Mononuclear Cells Research Articles

Conjugated linoleic acid (CLA) modulates bovine peripheral blood mononuclear cells (PBMC) proteome in vitro

Conjugated linoleic acid (CLA) is a group of natural isomers of the n-6 polyunsaturated fatty acid (PUFA) linoleic acid, exerting biological effects on cow physiology. This study assessed the impact of the mixture 50:50 (vol:vol) of CLA isomers (cis-9, trans-11 and trans-10, cis-12) on bovine peripheral blood mononuclear cells (PBMC) proteome, identifying 1608 quantifiable proteins. A supervised multivariate statistical analysis, sparse variant partial least squares – discriminant analysis (sPLS-DA) for paired data identified 407 discriminant proteins (DP), allowing the clustering between the CLA and controls. The ProteINSIDE workflow found that DP with higher abundance in the CLA group included proteins related to innate immune defenses (PLIN2, CD36, C3, C4, and AGP), with antiapoptotic (SERPINF2 and ITIH4) and antioxidant effects (HMOX1). These results demonstrated that CLA modulates the bovine PBMC proteome, supports the antiapoptotic and immunomodulatory effects observed in previous in vitro studies on bovine PBMC, and suggests a cytoprotective role against oxidative stress. SignificanceIn this study, we report for the first time that the mixture 50:50 (vol:vol) of cis-9, trans-11, and trans-10, cis-12-CLA isomers modulates the bovine PBMC proteome. Our results support the immunomodulatory and antiapoptotic effects observed in bovine PBMC in vitro. In addition, the present study proposes a cytoprotective role of CLA mixture against oxidative stress. We suggest a molecular signature of CLA treatment based on combining a multivariate sparse discriminant analysis and a clustering method. This demonstrates the great value of sPLS-DA as an alternative option to identify discriminant proteins with relevant biological significance.

Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

Dawn-to-dusk dry fasting induces anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic proteome in peripheral blood mononuclear cells in subjects with metabolic syndrome

BackgroundMetabolic syndrome characterized by abdominal obesity, high blood pressure, elevated fasting glucose and triglyceride levels and low high-density lipoprotein cholesterol level is associated with pro-inflammatory state, increased risk for atherosclerosis, and multiple cancers. Our previous results on subjects with metabolic syndrome showed that 4-week dawn-to-dusk (sunset) dry fasting resulted in significant changes in the serum proteome and improvement in several metabolic risk factors. Peripheral blood mononuclear cells (PBMC) proteomics is a powerful tool that can provide mechanistic insights into how dawn-to-dusk dry fasting affects protein expression in metabolic pathways at cellular level. In this study, we determined whether dawn-to-dusk dry fasting would induce favorable changes in PBMC proteome in subjects with metabolic syndrome, similar to the changes induced by dawn-to-dusk dry fasting in the same subjects' serum proteome. MethodsWe conducted a prospective study on subjects with metabolic syndrome and collected blood specimens before 4-week dawn-to-dusk dry fasting, at the end of 4-week dawn-to-dusk dry fasting, and one week after 4-week dawn-to-dusk dry fasting. We performed untargeted proteomics using nano ultra-high performance liquid chromatography-tandem mass spectrometry to assess the impact of 4-week dawn-to-dusk dry fasting on PBMC proteome. ResultsThere were 14 subjects with metabolic syndrome with a mean age of 59 who fasted from dawn to dusk (strict dry fasting without any liquid or food intake) for more than 14 h daily for 29 days. The quantitative proteome analysis showed that apolipoprotein B (APOB) gene protein products (GP) levels were downregulated and had the most statistical significance of the observed difference at the end of 4-week dawn-to-dusk dry fasting (P = 0.008) and one week after 4-week dawn-to-dusk dry fasting (P = 0.0004) compared with the levels before 4-week dawn-to-dusk dry fasting. The comparison between GP levels before and at the end of 4-week dawn-to-dusk dry fasting showed an alteration in the expression of genes associated with lipid and atherosclerosis pathway (P = 6.014e-4) and C-type lectin receptor signaling pathway (P = 1.064e-5). The genes that were differentially expressed in the lipid and atherosclerosis pathway were APOB (P = 0.008), CD36 (P = 0.040), CALM1, CALM2, CALM3 (P = 0.015), and HSPA8 (P = 0.047). One of the differentially expressed genes in the C-type lectin receptor signaling pathway was lymphocyte-specific protein 1 (LSP1), which showed an average of 19-fold increase at the end of 4-week dawn-to-dusk dry fasting compared with the GP levels before fasting (P = 0.004). Several GPs associated with tumor-suppressor effect (TUBB4B, LSP1, ACTR3B) were upregulated, and GPs associated with tumor-promoter effect (CD36, CALM1, CALM2, CALM3, FLOT2, PPIF) were downregulated at the end of 4-week dawn-to-dusk dry fasting or one week after 4-week dawn-to-dusk dry fasting compared with the GP levels before 4-week dawn-to-dusk dry fasting. ConclusionBased on our results, we conclude that in subjects with metabolic syndrome, 4-week dawn-to-dusk dry fasting induced anti-atherosclerotic, anti-inflammatory, and anti-tumorigenic PMBC proteome. Randomized, controlled clinical trials are needed to further investigate the effect of dawn-to-dusk dry fasting on subjects with chronic metabolic diseases and metabolic syndrome-induced cancers.

Human peripheral blood mononuclear cells: A review of recent proteomic applications.

Human peripheral blood mononuclear cells (PBMCs) represent a sentinel blood sample which reacts to different pathophysiological stimuli in the form of immunological responses/immunophenotypic changes. The study of molecular content of PBMCs can provide better understanding of immune processes giving the possibility of monitoring the health conditions of the host organism. Proteomic analysis of PBMCs can achieve mentioned goal as important immune-related biomarkers are easily accessible for analysis. PBMCs have been gaining attention in different research areas including preclinical or clinical investigations. In this review, recent applications of proteomic analysis of PBMCs are described and discussed. Approaches are divided based on different proteomic workflows such as in-gel, in-solution and on-filter modes. The effect of various diseases such as autoimmune, cancer, neurodegenerative, viral, metabolic, and various immune stimulations such as radiation, vaccine, corticosteroids over PBMCs proteome, are described with emphasis on promising protein biomarker candidates.

Identification of proteins associated with development of psoriatic arthritis in peripheral blood mononuclear cells: a quantitative iTRAQ-based proteomics study

BackgroundBiomarkers for distinguishing psoriatic arthritis (PsA) from psoriasis without arthritis (PsO) are still lacking.MethodsWe applied isobaric tags for relative and absolute quantification (iTRAQ) and LC–MS/MS to analyze the proteome profile of peripheral blood mononuclear cells (PBMCs) collected from patients with PsO, patients with PsA, and healthy controls. Bioinformatics analysis and western blotting were performed to identify and validate differentially expressed proteins.ResultsWe identified 389, 199, 291, and 60 significantly differentially expressed proteins (adj.p < 0.05) in the comparison of all psoriatic patients versus healthy controls, PsO group versus healthy controls, PsA group versus healthy controls, and PsA group versus PsO group, respectively. Among these proteins, 14 proteins may represent promising biomarkers for PsA: SIRT2, NAA50, ARF6, ADPRHL2, SF3B6, SH3KBP1, UBA3, SCP2, RPS5, NUDT5, NCBP1, SYNE1, NDUFB7, HTATSF1. Furthermore, western blotting confirmed that SIRT2 expression was significantly higher in PBMCs from PsA patients than PsO and healthy controls, and was negatively correlated with the phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK; p = 0.006, r = − 0.582).ConclusionsThis pilot study provided a broad characterization of the proteome of PBMCs in PsA as compared to PsO and healthy controls, which may help to provide prospective strategies for PsA diagnosis.

A new strategy to uncover fragile X proteomic biomarkers using the nascent proteome of peripheral blood mononuclear cells (PBMCs)

Fragile X syndrome (FXS) is the most prevalent inherited cause of intellectual disabilities and autism spectrum disorders. FXS result from the loss of expression of the FMRP protein, an RNA-binding protein that regulates the expression of key synaptic effectors. FXS is also characterized by a wide array of behavioural, cognitive and metabolic impairments. The severity and penetrance of those comorbidities are extremely variable, meaning that a considerable phenotypic heterogeneity is found among fragile X individuals. Unfortunately, clinicians currently have no tools at their disposal to assay a patient prognosis upon diagnosis. Since the absence of FMRP was repeatedly associated with an aberrant protein synthesis, we decided to study the nascent proteome in order to screen for potential proteomic biomarkers of FXS. We used a BONCAT (Biorthogonal Non-canonical Amino Acids Tagging) method coupled to label-free mass spectrometry to purify and quantify nascent proteins of peripheral blood mononuclear cells (PBMCs) from 7 fragile X male patients and 7 age-matched controls. The proteomic analysis identified several proteins which were either up or downregulated in PBMCs from FXS individuals. Eleven of those proteins were considered as potential biomarkers, of which 5 were further validated by Western blot. The gene ontology enrichment analysis highlighted molecular pathways that may contribute to FXS physiopathology. Our results suggest that the nascent proteome of PBMCs is well suited for the discovery of FXS biomarkers.

Proteomic analysis of peripheral blood mononuclear cells and inflammatory status in postpartum dairy cows supplemented with different sources of omega-3 fatty acids

We examined the effects of dietary n-3 fatty acids on the proteome of peripheral blood mononuclear cells (PBMC) in transition dairy cows. Forty-two dry cows were divided into three groups supplemented with: saturated fat (CTL); flaxseed oil (FLX); or fish oil (FO). PBMC were collected from five cows per group at week 1 postpartum for proteomic analysis. The n-3 fatty acid content in plasma and PBMC was higher in FLX and FO than in CTL cows. In PBMC, 3807 proteins were quantified and 44, 42 and 65 were differently abundant in FLX vs. CTL, FO vs. CTL and FLX vs. FO, respectively. In FLX vs. CTL, the abundance of the p65-subunit-of-transcription-factor NF-κB was higher, whereas albumin, C4b-binding protein and complement factor H levels were lower. In FLX vs. FO, complement factors B and H and hemopexin were higher. The top canonical pathway enriched in FLX compared to other groups was acute-phase-response signaling. The percentage of CD25+ blood cells was lower in FLX and FO at 1 week postpartum, and gene expression of NF-κB in white blood cells was lower in FLX than in CTL. Dietary sources of n-3 fatty acids differentially affected the proteome of PBMC, possibly altering the inflammatory status. SignificanceThe transition dairy cow experiences a variable degree of systemic subacute inflammation, and proteomics of peripheral blood mononuclear cells (PBMC) may contribute to obtain insight into this process. Omega-3 fatty acids can moderate the immunological effect, and therefore we examined the effects of these fatty acids from flaxseed (FLX) or fish oils (FO) on the proteome of PBMC at week 1 postpartum. More than 3800 proteins were quantified, and in cows supplemented with FLX, enrichment of the acute-phase-signaling and complement systems were apparent in the PBMC compared to CTL and FO PBMC. This information may be useful to further explore the mechanism by which dietary omega-3 fatty acids affect the immune system in postpartum dairy cows.

Proteomic Analysis of Peripheral Blood Mononuclear Cells Following a Single High Fat High Carbohydrate Meal Challenge

ObjectivesAn individual’s ability to adapt to a dynamic environment is termed ‘phenotypic flexibility’ and can be weakened by chronic stress, leading to dysregulation of normal homeostatic processes. A long-term high energy and high fat high carbohydrate (HFHC) diet increases risk of metabolic diseases, however, the acute effect of a single HFHC meal on disturbances in metabolic homeostasis is less understood. In this study, we aimed to characterise the effect of a single HFHC meal on the postprandial proteome of peripheral blood mononuclear cells (PBMCs). MethodsTwelve healthy men (22.7 ± 3.4 years, BMI of 22.2 ± 1.5 kg/m2) were recruited. Participants consumed a HFHC milkshake after fasting overnight. The meal contained 16 g of carbohydrate and 15 g of fat per 100 g and had a total energy content of 64 kJ/kg body weight. PBMCs were collected at fasting, 3 and 6 hours following the meal. The proteome was measured via LC-MS/MS using untargeted label-free quantification. Differentially expressed proteins were identified with a modified paired t test (limma, R v.4.0.3). Gene ontology analysis was performed with ClueGO v2.5.7 in Cytoscape v3.8.2. ResultsAfter filtering to include only high confident proteins consistently detected in each condition, 6007 proteins were identified and included in analysis. Changes in protein expression relative to fasting were observed at 3 hours (143 proteins) and 6 hours (210 proteins) post meal (fold change > ±1.2, p < 0.05). The most highly altered biological processes observed at each timepoint were ‘zymogen activation’ (GO:0,031,638, 31.25% of groups at 3 hours) and ‘ribosome biogenesis’ (GO:0,042,254, 19.15% of groups at 6 hours). ConclusionsThese results reveal adaptations that occur in the PBMC proteome in response to a single HFHC meal. Changes to proenzyme activation shows a general response in PBMCs to meal-induced changes in the cellular environment, whilst regulation of ribosome synthesis pathways may reflect changes in energy regulation pathways caused by increased nutrient availability. This study characterises the PBMC response in healthy men, and provides a useful reference for future studies that investigate how PBMCs lose the ability to maintain homeostasis following a HFHC meal. Funding SourcesMonash University, Melbourne, Australia

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.

Profiling of the Peripheral Blood Mononuclear Cell Proteome in Schizophrenia and Mood Disorders for the Discovery of Discriminatory Biomarkers: A Proof-of-Concept Study

Introduction: Current diagnoses in psychiatry are solely based on the evaluation of clinical presentation by the treating psychiatrist. This results in a high percentage of misdiagnosis and consequential inefficient treatment; especially regarding major depressive disorder (MDD), depression in the context of bipolar disorder (BD-D), bipolar disorder with manic symptoms (BD-M), and psychosis in the context of schizophrenia (SZ). Objective biomarkers allowing for accurate discriminatory diagnostics are therefore urgently needed. Methods: Peripheral blood mononuclear cell (PBMC) proteomes of patients with MDD (n = 5) , BD-D (n = 3), BD-M (n = 4), and SZ (n = 4), and also of healthy controls (HC; n = 6) were analyzed by state-of-the-art mass spectrometry. Proteins with a differential expression of a >2 standard deviation (SD) expression fold change from that of the HC and between either MDD versus BD-D or BD-M versus SZ were subsequently identified as potential discriminatory biomarkers. Results: In total, 4,271 individual proteins were retrieved from the HC. Of these, about 2,800 were detected in all patient and HC samples. For objective discrimination between MDD and BD-D, 66 candidate biomarkers were found. In parallel, 72 proteins might harbor a biomarker capacity for differential diagnostics of BD-M and SZ. A single biomarker was contraregulated versus HC in each pair of comparisons. Discussion: With this work, we provide a register of candidate biomarkers with the potential to objectively discriminate MDD from BD-D, and BD-M from SZ. Although concerning a proof-of-concept study with limited sample size, these data provide a stepping-stone for follow-up research on the validation of the true discriminatory potential and feasibility of clinical implementation of the discovered biomarker candidates.

Peripheral blood mononuclear cell proteome profile in Behçet’s syndrome

Behçet's syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Investigation of proteome profiles of disease specific cells facilitates our understanding of the processes and related molecular pathways, especially in disorders like BS with complex inheritance pattern and clinical heterogeneity. In the current study, we evaluated the peripheral blood mononuclear cells (PBMCs) proteome of 59 patients with BS (33 in active and 26 in inactive phases) and of 28 healthy controls using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed protein spots with at least twofold and/or statistically significant change (p ≤ 0.05) between active BS vs inactive BS, and also active BS vs healthy controls were identified by mass spectrometry (MALDI-TOF/TOF). Bioinformatic analyses revealed 16 differentially expressed proteins (12 of them in active vs inactive BS comparison, whereas 11 of them for active BS vs healthy control comparison) belonging to glycolysis, cytoskeleton organization, protein folding, and regulation of blood coagulation pathways. Stathmin (active BS vs inactive BS; fourfold, active BS vs healthy control; 4.7-fold) and WD repeat-containing protein-1 (active BS vs inactive BS; 2.7-fold, active BS vs healthy control; 2.7-fold), which are cytoskeleton-related proteins, were found to be lower in active patients compared to inactive patients and healthy control. Decreased levels of calreticulin (active BS vs inactive BS; 1.29-fold) and heat shock 70kDa protein 8 (active BS vs healthy control; 1.5-fold) which are involved in protein folding and endoplasmic reticulum (ER) stress process, were observed in patients with active phase of BS. Down-regulation of protein folding and ER stress process proteins in BS patients may further support the involvement of ER stress in BS.

Discovering Protein Biomarkers from Clinical Peripheral Blood Mononuclear Cells Using Data-Independent Acquisition Mass Spectrometry.

Global proteomics analyses are traditionally performed in data-dependent acquisition (DDA) mode, which results in inadequate reproducibility across large sample cohorts due to the under-sampling inherent to shotgun proteomics. Recently, data-independent acquisition (DIA) strategies were introduced to allow reproducible detection and quantification of thousands of proteins with consistent sensitivity across samples. Here, we present an approach to analyze changes to the protein network in human peripheral blood mononuclear cells (PBMCs) from clinical blood samples, using DIA as a unique platform for biomarker discovery. We describe how to generate spectral PBMC proteome libraries by applying peptide fractionation followed by DDA analysis, and then how to apply DIA methods to PBMC samples from individual patients using a high-resolution Orbitrap Fusion mass spectrometer.

A Comprehensive Proteome Analysis of Peripheral Blood Mononuclear Cells (PBMCs) to Identify Candidate Biomarkers of Pancreatic Cancer.

Pancreatic cancer (PC) is currently the fourth leading cause of cancer-related mortality worldwide. Peripheral blood mononuclear cells (PBMCs) is a subpopulation of accessible and functional immune cells. Comparative analysis of the proteome of PBMCs can help us elucidate the mechanism of disease and find potential biomarkers for diagnosis. PBMCs were collected from healthy individuals, patients with benign diseases, and pancreatic cancer. iTRAQ-2DLC-MS/MS and SWATH methodologies were applied to make a comparative proteomics analysis of PBMCs. A total of 3,357 proteins with a false discovery rate (FDR) <1% were identified, of which 114 proteins were found dysregulated in the PC group. An extensive SWATH library was constructed which showed a potential application for large scale clinical sample analysis. A PBMCs proteome with extensive protein representation was achieved, which will potentially allow the identification of novel biomarkers for PC.

Effects on pig immunophysiology, PBMC proteome and brain neurotransmitters caused by group mixing stress and human-animal relationship.

Peripheral blood mononuclear cells (PBMC) are an interesting sample for searching for biomarkers with proteomic techniques because they are easy to obtain and do not contain highly abundant, potentially masking proteins. Two groups of pigs (n = 56) were subjected to mixing under farm conditions and afterwards subjected to different management treatments: negative handling (NH) and positive handling (PH). Serum and PBMC samples were collected at the beginning of the experiment one week after mixing (t0) and after two months of different handling (t2). Brain areas were collected after slaughter and neurotransmitters quantified by HPLC. Hair cortisol and serum acute phase proteins decreased and serum glutathione peroxidase increased at t2, indicating a lower degree of stress at t2 after adaptation to the farm. Differential gel electrophoresis (DIGE) was applied to study the effects of time and treatment on the PBMC proteome. A total of 54 differentially expressed proteins were identified, which were involved in immune system modulation, cell adhesion and motility, gene expression, splicing and translation, protein degradation and folding, oxidative stress and metabolism. Thirty-seven protein spots were up-regulated at t2 versus t0 whereas 27 were down-regulated. Many of the identified proteins share the characteristic of being potentially up or down-regulated by cortisol, indicating that changes in protein abundance between t0 and t2 are, at least in part, consequence of lower stress upon adaptation to the farm conditions after group mixing. Only slight changes in brain neurotransmitters and PBMC oxidative stress markers were observed. In conclusion, the variation in hair cortisol and serum APPs as well as the careful analysis of the identified proteins indicate that changes in protein composition in PBMC throughout time is mainly due to a decrease in the stress status of the individuals, following accommodation to the farm and the new group.

In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

Interindividual Variation in the Proteome of Human Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMCs) are main actors in inflammatory processes and linked to many diseases, including rheumatoid arthritis, atherosclerosis, asthma, HIV and cancer. Moreover, they seem an interesting ‘surrogate tissue’ that can be used in biomarker discovery. In order to get a good experimental design for quantitative expression studies, the knowledge of the interindividual variation is an essential part. Therefore, PBMCs were isolated from 24 healthy volunteers (15 males, 9 females, ages 63–86) with no clinical signs of inflammation. The extracted proteins were separated using the two dimensional difference in gel electrophoresis technology (2D-DIGE), and the gel images were processed with the DeCyder 2D software. Protein spots present in at least 22 out of 24 healthy volunteers were selected for further statistical analysis. Determination of the coefficient of variation (CV) of the normalized spot volume values of these proteins, reveals that the total variation of the PBMC proteome varies between 12,99% to 148,45%, with a mean value of 28%. A supplemental look at the causes of technical variation showed that the isolation of PBMCs from whole blood is the factor which influences the experimental variance the most. This isolation should be handled with extra care and an additional washing step would be beneficial. Knowing the extent of variation, we show that at least 10 independent samples per group are needed to obtain statistical powerful data. This study demonstrates the importance of considering variance of a human population for a good experimental design for future protein profiling or biomarker studies.

Proteomic analysis of blood cells in fish exposed to chemotherapeutics: Evidence for long term effects

Proteomics technology are increasingly used in ecotoxicological studies to characterize and monitor biomarkers of exposure. The present study aims at identifying long term effects of malachite green (MG) exposure on the proteome of peripheral blood mononuclear cells (PBMC) from the Asian catfish, Pangasianodon hypophthalmus. A common (0.1 ppm) concentration for therapeutic treatment was applied twice with a 72 h interval. PBMC were collected directly at the end of the second bath of MG (T1) and after 1 month of decontamination (T2). Analytical 2D-DIGE gels were run and a total of 2551±364 spots were matched. Among them, MG induced significant changes in abundance of 116 spots with no recovery after one month of decontamination. Using LC-MS/MS and considering single identification per spot, we could identify 25 different proteins. Additionally, MG residues were measured in muscle and in blood indicating that leuco-MG has almost totally disappeared after one month of decontamination. This work highlights long term effects of MG treatment on the PBMC proteome from fish intended for human consumption.

2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting

BackgroundProteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation.MethodsBoth a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers.ResultsBetween-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells.ConclusionsIdentification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.

Changes in the porcine peripheral blood mononuclear cell proteome induced by infection with highly virulent classical swine fever virus

Leukopenia and immunosuppression are characteristic clinical manifestations of classical swine fever and peripheral blood mononuclear cells (PBMCs) are major targets of classical swine fever virus. To investigate proteomic expression changes in swine PBMCs during lethal CSFV infection, proteins of PBMCs from five lethally CSFV-infected pigs were resolved by two-dimensional electrophoresis followed by mass spectrometry. Quantitative intensity analysis revealed that 66 protein spots showed altered expression, 44 of which were identified as 34 unique proteins by MALDI-TOF-MS/MS. Cellular functions of these proteins included cytoskeletal, energy metabolism, protein translation and processing, antioxidative stress, heat shock and blood clotting. Western blot analysis confirmed the upregulation of annexin A1 and downregulation of cofilin. Identification of these changed levels of expression provides an understanding at the molecular level of the response of in vivo target cells to CSFV infection and of the pathogenic mechanisms of leukopenia and immunosuppression induced by the virus.

The human peripheral blood mononuclear cell proteome responds to a dietary flaxseed‐intervention and proteins identified suggest a protective effect in atherosclerosis

Flaxseed is one of the richest sources of lignans that are converted to enterolactone by the intestinal microflora. Enterolactone has been suggested to be the prime active compound mediating atherosclerosis-protective effects that were shown for flaxseed. The effects of a 1-wk intervention with 0.4 g of flaxseed/kg body weight per day on enterolactone plasma levels in seven healthy men revealed that all participants (PAs) responded with enhanced enterolactone plasma levels. Proteome analysis of peripheral blood mononuclear cells (PBMC) from donors before, during, and after the intervention showed that flaxseed consumption affected significantly the steady-state levels of 16 proteins of which four were altered in a similar manner when blood mononuclear cells were exposed ex vivo to enterolactone. Enhanced levels of peroxiredoxin and reduced levels of the long-chain fatty acid beta-oxidation multienzyme complex may be taken as indicators of a reduced oxidative stress whereas reduced levels of glycoprotein IIIa/II could indicate improved protection from thrombotic and inflammatory processes. In conclusion, the blood mononuclear cell proteome responds to dietary flaxseed intake with changes in a number of atherosclerosis-relevant proteins that may be taken as biomarkers of exposure and some of these changes observed can be attributed to the action of the lignan metabolite enterolactone.