How can platelet proteomics best be used to interrogate disease?

Various modifications of proteins and the resulting proteoforms of a protein can associate with many diseases and are also significantly involved in the rapid regulation of hemostasis and thrombosis. For example, the release of prostacyclin from the intact endothelium and the consequent following phosphorylation of VASP in platelets is a post-translational regulation to keep them in a quiescent state. In Alzheimer's disease, proteoforms arise from the altered cleavage of the amyloid precursor protein, which finally causes amyloid plaques in the brain. This changed processing of the amyloid precursor protein can also be detected in platelets, making them an attractive source of biomarkers for this neurodegenerative disease. Age-related or prothrombotic disorders can have multiple origins, including genomic, transcriptional, and translational factors, which together can be mapped at the proteome level. Hence, recording these dynamic protein changes under physiological and pathophysiological conditions is paramount in platelet proteomics. To effectively study diseases through platelet proteomics, it is crucial to consider platelets' primary regulatory mechanism and thoroughly evaluate the disparities between the two leading proteomics technologies, top-down and bottom-up approaches. This commentary provides insights into the differences between these two technologies, which are particularly noticeable in detecting the different proteoforms of a protein.

What is the context?The repertoire of all proteins in a biological sample is the proteome. Proteomics refers to different biochemical technologies that detect and quantify the proteins in a biological sample, such as platelets. If proteome analyses are carried out on a representative number of samples from a specific patient group and compared to a matched control group, disease-dependent changes in proteins can be found that indicate unknown causes of the disease or also be used as biomarkers for diagnosis and prognosis. It is also essential to consider that the proteins in biological samples can occur in various variations, the proteoforms. These proteoforms of a protein can arise, for example, through genetically-based variations or regulatory post-translational protein modifications.What's new?There are two fundamentally different methods in proteomics technology: top-down and bottom-up. For bottom-up proteomics, the proteins must be digested into peptides for technical reasons, whereas top-down proteomics analyzes intact proteins. These different sample processing steps significantly impact the resulting data set. This is particularly crucial for platelet proteomics studies, as primary hemostasis is mainly carried out through post-translational modifications of proteins, resulting in various proteoforms that regulate platelet reactivity and thrombus formation.What's the impact?Bottom-up proteomics can quickly and automatically identify an extensive repertoire of proteins from a platelet sample. This is much more cumbersome with top-down proteomics. In contrast, here, various intact proteoforms of intact proteins can be unbiasedly detected and directly quantified, which is particularly important for examining the global proteome of platelets in clinical samples. This qualitative and quantitative relative assignment to the respective proteoforms of a protein is not possible in bottom-up proteomics.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Platelet Phenotype Analysis of COVID-19 Patients Reveals Progressive Changes in the Activation of Integrin αIIbß3, F13A1, the SARS-CoV-2 Target EIF4A1 and Annexin A5.

Background: The fatal consequences of an infection with severe acute respiratory syndrome coronavirus 2 are not only caused by severe pneumonia, but also by thrombosis. Platelets are important regulators of thrombosis, but their involvement in the pathogenesis of COVID-19 is largely unknown. The aim of this study was to determine their functional and biochemical profile in patients with COVID-19 in dependence of mortality within 5-days after hospitalization. Methods: The COVID-19-related platelet phenotype was examined by analyzing their basal activation state via integrin αIIbß3 activation using flow cytometry and the proteome by unbiased two-dimensional differential in-gel fluorescence electrophoresis. In total we monitored 98 surviving and 12 non-surviving COVID-19 patients over 5 days of hospital stay and compared them to healthy controls (n = 12). Results: Over the observation period the level of basal αIIbß3 activation on platelets from non-surviving COVID-19 patients decreased compared to survivors. In line with this finding, proteomic analysis revealed a decrease in the total amount of integrin αIIb (ITGA2B), a subunit of αIIbß3, in COVID-19 patients compared to healthy controls; the decline was even more pronounced for the non-survivors. Consumption of the fibrin-stabilizing factor coagulation factor XIIIA (F13A1) was higher in platelets from COVID-19 patients and tended to be higher in non-survivors; plasma concentrations of the latter also differed significantly. Depending on COVID-19 disease status and mortality, increased amounts of annexin A5 (ANXA5), eukaryotic initiation factor 4A-I (EIF4A1), and transaldolase (TALDO1) were found in the platelet proteome and also correlated with the nasopharyngeal viral load. Dysregulation of these proteins may play a role for virus replication. ANXA5 has also been identified as an autoantigen of the antiphospholipid syndrome, which is common in COVID-19 patients. Finally, the levels of two different protein disulfide isomerases, P4HB and PDIA6, which support thrombosis, were increased in the platelets of COVID-19 patients. Conclusion: Platelets from COVID-19 patients showed significant changes in the activation phenotype, in the processing of the final coagulation factor F13A1 and the phospholipid-binding protein ANXA5 compared to healthy subjects. Additionally, these results demonstrate specific alterations in platelets during COVID-19, which are significantly linked to fatal outcome.

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin-α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Altered platelet proteome in lupus anticoagulant (LA)-positive patients-protein disulfide isomerase and NETosis as new players in LA-related thrombosis.

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE-) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.

Platelet PI3K Modulates Innate Leukocyte Extravasation during Acid-Induced Acute Lung Inflammation.

INTRODUCTION: Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. OBJECTIVE: Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. MATERIALS AND METHODS: We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet-leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. RESULTS: In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. CONCLUSION: Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

Gender-related increase of tropomyosin-1 abundance in platelets of Alzheimer's disease and mild cognitive impairment patients.

The incidence of Alzheimer's disease (AD) is higher in elderly women than in men. The molecular background of this gender-related risk, however, is largely unknown. In a previous proteomics study, we identified significantly elevated levels of monoamine oxidase-B and tropomyosin-1 in AD patients, together with significant changes of the genetic AD risk factors apolipoprotein E4 (APOE4) and glutathione S-transferase omega 1 (GSTO1), in platelets - a promising source for AD blood biomarkers. The present study aimed to investigate the gender-specificity as well as the disease-stage dependency of these biomarkers in AD patients and those with mild cognitive impairment (MCI). Tropomyosin-1 and monoamine oxidase-B protein levels were quantified by 2-D DIGE and 1-D Western blotting. Here, for the first time, we revealed a significant increase of 38&39kDa tropomyosin-1 protein levels in female but not male AD (+56%; p=0.008) and MCI patients (+46%; p=0.041) measured by 1-D WB. In contrast, levels of monoamine oxidase-B were, independently of gender, elevated in AD patients (+52%; p=0.009) but unaltered in MCI compared to control subjects. Moreover, we confirmed that APOE4-positive females are at a higher risk (OR=18.7; p=9.7E-09) of developing AD compared to APOE4-positive males (OR=6.5; p=5.9E-04). No gender-related effects were observed for GSTO1. SIGNIFICANCE: Platelet tropomyosin-1 constitutes a gender-related and stage-dependent protein in cognitive impairment. In contrast, platelet monoamine oxidase-B, frequently described to be increased in platelets and brains of AD patients, shows a gender-independent but stage-related increase since it is unaltered in MCI subjects. A blood biomarker test for this preceding stage of AD that considers gender-specificity is not yet available. The newly described AD-related platelet protein profiles might refine and facilitate routine diagnosis and enable early as well as tailored interventions.

Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods.

BACKGROUND: Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of the E4 allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous. METHODS: Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels. RESULTS: Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for the E4 allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity. CONCLUSIONS: This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual's APOE4 status directly from plasma. The E4 genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer's disease. The biochip's potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-based APOE genotyping and may deliver additional protein-specific information on a variety of diseases in the future.

Careful neuropsychological testing reveals a novel genetic marker, GSTO1*C, linked to the pre-stage of Alzheimer's disease.

Approximately 30 million people currently suffer from late-onset Alzheimer's disease (LOAD) worldwide. Twin studies demonstrated that 60 to 80% of LOAD is genetically determined, 20% of which remaining unassigned. This case-control study included 118 cognitively healthy controls, 52 patients with mild cognitive impairment (MCI; the pre-stage of LOAD) and 71 LOAD patients. The participants were genotyped for the genetic LOAD marker apolipoprotein E4 (APOE4) and the single-nucleotide polymorphism rs4925 in glutathione S-transferase omega-1 (GSTO1). Additive logistic regression showed a novel, statistically significant association of the major allele GSTO1*C with MCI (OR1.9; p = 0.032). However, identification of significant SNP-disease relations required well-defined study groups. When classifying participants solely by the short Mini Mental State examination (MMSE), the associations of GSTO1*C and the reference marker APOE4 with MCI were cancelled. Moreover, even identifying only the control group by MMSE nullified a statistically significant association (OR1.8; p = 0.045) between GSTO1*C and LOAD. In contrast, these statistical relations were retained when the detailed Consortium to Establish a Registry for Alzheimer's Disease (CERAD-Plus) test battery was used. Hence, besides proposing rs4925 as a genetic marker for cognitive impairment, this work also emphasized the importance of carefully characterized controls in addition to well-diagnosed patients in case-control studies.

Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration.

We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

Proteome analysis of testis from infertile protein C inhibitor-deficient mice reveals novel changes in serpin processing and prostaglandin metabolism.

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.

A platelet protein biochip rapidly detects an Alzheimer's disease-specific phenotype.

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.

Autocrine secretion of 15d-PGJ2 mediates simvastatin-induced apoptotic burst in human metastatic melanoma cells.

BACKGROUND AND PURPOSE: Despite new therapeutic approaches, metastatic melanomas still have a poor prognosis. Statins reduce low-density lipoprotein cholesterol and exert anti-inflammatory and anti-proliferative actions. We have recently shown that simvastatin triggers an apoptotic burst in human metastatic melanoma cells by the synthesis of an autocrine factor. EXPERIMENTAL APPROACH: The current in vitro study was performed in human metastatic melanoma cell lines (A375, 518a2) and primary human melanocytes and melanoma cells. The secretome of simvastatin-stressed cells was analysed with two-dimensional difference gel electrophoresis and MS. The signalling pathways involved were analysed at the protein and mRNA level using pharmacological approaches and siRNA technology. KEY RESULTS: Simvastatin was shown to activate a stress cascade, leading to the synthesis of 15-deoxy-12,14-PGJ2 (15d-PGJ2 ), in a p38- and COX-2-dependent manner. Significant concentrations of 15d-PGJ2 were reached in the medium of melanoma cells, which were sufficient to activate caspase 8 and the mitochondrial pathway of apoptosis. Inhibition of lipocalin-type PGD synthase, a key enzyme for 15d-PGJ2 synthesis, abolished the apoptotic effect of simvastatin. Moreover, 15d-PGJ2 was shown to bind to the fatty acid-binding protein 5 (FABP5), which was up-regulated and predominantly detected in the secretome of simvastatin-stressed cells. Knockdown of FABP5 abolished simvastatin-induced activation of PPAR-γ and amplified the apoptotic response. CONCLUSIONS AND IMPLICATIONS: We characterized simvastatin-induced activation of the 15d-PGJ2 /FABP5 signalling cascades, which triggered an apoptotic burst in melanoma cells but did not affect primary human melanocytes. These data support the rationale for the pharmacological targeting of 15d-PGJ2 in metastatic melanoma.

Platelets, a reliable source for peripheral Alzheimer's disease biomarkers?

Peripheral biomarkers play an indispensable role in quick and reliable diagnoses of any kind of disease. With the population ageing, the number of people suffering from age-related diseases is expected to rise dramatically over the coming decades. In particular, all types of cognitive deficits, such as Alzheimer's disease, will increase. Alzheimer's disease is characterised mainly by coexistence of amyloid plaques and neurofibrillary tangles in brain. Reliable identification of such molecular characteristics antemortem, however, is problematic due to restricted availability of appropriate sample material and definitive diagnosis is only possible postmortem. Currently, the best molecular biomarkers available for antemortem diagnosis originate from cerebrospinal fluid. Though, this is not convenient for routine diagnosis because of the required invasive lumbar puncture. As a consequence, there is a growing demand for additional peripheral biomarkers in a more readily accessible sample material. Blood platelets, due to shared biochemical properties with neurons, can constitute an attractive alternative as discussed here. This review summarises potential platelet Alzheimer's disease biomarkers, their role, implication, and alteration in the disease. For easy comparison of their performance, the Hedge effect size was calculated whenever data were available.

Identification and validation of platelet low biological variation proteins, superior to GAPDH, actin and tubulin, as tools in clinical proteomics.

Accurate biomarker quantification requires carefully chosen normalisation procedures. When single proteins are used as loading controls (LCs), it is crucial that their expressional stability must be known. Platelets are an important biomarker source, especially for neurological diseases. We performed a systematical analysis of the platelet proteome to identify proteins suitable as LCs, using the 2-D DIGE system. We first screened a healthy population (n=137), aged between 18 and 104years, to find proteins with small coefficients of total variation (CVtot), herein termed low biological variation proteins (LBVP). Thereafter, expressional stability was verified in 101 patients suffering from Alzheimer's- (AD), Parkinson's- disease, vascular dementia or schizophrenia. Interestingly, traditional LCs such as tubulin beta-1 and GAPDH, were not found amongst LBVP. The least variable protein, calculated over all 238 individuals, was 14-3-3 gamma, with a CVtot of 9.3%, showing no gender, age or disease dependency. The normalisation capability of 14-3-3 gamma was superior to traditional LC in quantifying Western blot signals of the platelet AD-biomarker Monoamine Oxidase B of patient versus controls. Similar results were obtained with HepG2 cells, treated in vitro with DNA-methyltransferase inhibitor 5-aza-2'deoxicytidine. Finally, we provide a list of alternative normalisation candidates for accurate biomarker quantification. BIOLOGICAL SIGNIFICANCE: This paper suggests a considerable list of platelet proteins with a lower biological variation than well known "housekeeping" proteins like GAPDH and tubulin. Spot abundances of found proteins are middle ranged and unaffected by gender, age and certain diseases. Hence, listed proteins might be valuable normalisation candidates used additionally or alternatively. Platelet's least variable protein 14-3-3 gamma is validated as normalisation protein in platelet biomarker quantification. Furthermore 14-3-3 gamma is demonstrated to be also stable expressed by in HepG2, cells others than platelets, when treated by DNA methylation inhibitor.

A combined proteomic and genetic analysis of the highly variable platelet proteome: from plasmatic proteins and SNPs.

High biological variation in protein expression represents a major challenge in clinical proteomics. In a study based on 2D-DIGE, we found that the standardised abundance of only a few proteins varied by more than 50%. While some of the highest variable proteins in platelets of 52 healthy elderly were of plasmatic origin, such as albumin or haptoglobin, absence of several other high-abundant plasma proteins strongly suggests that plasma-derived proteins represent an integral part of the platelet proteome. Amongst the highly variable platelet-derived proteins, two spots were both identified as GSTO1 and assigned to either the wild-type or mutant isoform of SNP A140D. Remarkably, when the spots were considered within the respective genotype groups, their CV decreased to about the median variation. Albeit 2D-DIGE allowed correct genotyping, two individuals seemed to be GSTO1*A140 deficient. Probing 2D-Western blots with novel mAb, however, detected A140 protein as additional spot at pH 8.1, caused by the SNPs E155del and E208K. In contrast to previous studies, we show that GSTO1 protein is expressed in vivo, despite the deletion E155. Our data indicate that incorporation of exogenous proteins and genetic polymorphisms of endogenous proteins represent the main source of extreme biological variation in the platelet proteome.

How many spots with missing values can be tolerated in quantitative two-dimensional gel electrophoresis when applying univariate statistics?

Quantitative proteomic comparisons require a sufficient number of samples to reach an acceptable level of significance. But 2D gel electrophoresis commonly results in incomplete data sets due to spots with missing values reducing thereby the number of parallel measurements for individual proteins. Here we investigated how many missing values per spot can be tolerated. The number of spots in common between all gels was found to decrease with the number of parallel gels in a non-linear fashion. Increasing numbers of missing values were associated with a moderate increase in the quantitative variation of spot volumes. Based on the missing value pattern in 20 gels we performed an analysis of the multiple testing power for the hypothetical scenario of a comparative 2DE study with six or twelve parallel gels. The calculation considered the statistical power of the individual spot as well as the number of spots included in the analysis. The power increased with inclusion of spots with higher number of missing values and showed an optimum at a specific minimum number of spot replicates. The results suggest that proteins with missing values can be included in a univariate analysis as long as a sufficient number of parallel gels are made.

Comparative platelet proteome analysis reveals an increase of monoamine oxidase-B protein expression in Alzheimer's disease but not in non-demented Parkinson's disease patients.

Monoamine oxidase-B (Mao-B) catalysing the breakdown of the neurotransmitter dopamine, is known to be involved in the pathophysiology of Parkinson's (PD) and Alzheimer's disease (AD). Increased brain Mao-B activity is associated with AD. This alteration can also be seen in platelets, albeit the cause has hitherto remained elusive. To gain a deeper understanding of the etiology of AD, the platelet proteome was characterised, (2D DIGE pH6-9, including Mao-B) from 150 individuals: 34 AD, 13 vascular dementia, 15 non-demented PD patients, 49 matched controls, 18 oldest old and 21 young individuals. One significant change was noted after applying false discovery rate with the upregulation of the Mao-B expression (30% adjusted P value<0.001; effect size 1.31) in AD compared to age- and sex-matched controls. In contrast, Mao-B levels were unchanged in PD to matched controls. Western blot and mRNA analyses verified these findings. Moreover, Mao-B concentration correlated with age in the cognitive healthy individuals (r=0.53; P<0.001) and PD patients but not in those suffering from AD (r=-0.03; P=0.874). Mao-B activity correlated with the increased Mao-B protein expression in AD (r=0.81; P=0.016). We suggest that Mao-B platelet protein level may serve as a biomarker for age-related dementia, especially AD.

A proteomics study reveals a predominant change in MaoB expression in platelets of healthy volunteers after high protein meat diet: relationship to the methylation cycle.

Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP) diet on both cognitive performance and corresponding biochemical parameters. A randomised intervention study was conducted with 23 healthy males (aged 19-31 years) to investigate the effects of a usual (UP) versus a HP diet on cognitive function and on the platelet proteome a well-established model for neurons. The study individuals were assigned to either a UP diet (15% energy) or a HP diet (30% energy) for 3 weeks with controlled intake of food and beverages. Blood samples were taken along with measurements of cognitive functions at the beginning and at the end of the intervention period. Among 908 reproducibly studied platelet proteins only the level of monoamine oxidase B (MaoB), a neurotransmitter degrading enzyme, decreased by 26% significantly (adjusted P value < 0.05) due to the HP diet. In addition, we found a correlation (r = 0.477; P < 0.02) between the decrease of MaoB expression and the shortened reaction time (cognitive function) which is in accordance with reports that dementia patients show increased MaoB activity. Plasma vitamin B(12) concentration was increased by the HP diet and correlates inversely with platelet MaoB expression (r = -0.35; P < 0.02). Healthy young males on a HP diet showed improved cognitive function and counteract well-known dementia biomarkers such as platelet MaoB and components of the methylation cycle such as vitamin B(12) and homocysteine.

Effect of a high protein meat diet on muscle and cognitive functions: a randomised controlled dietary intervention trial in healthy men.

BACKGROUND: Recommendations to use other criteria than N-balance for defining protein requirements have been proposed. However, little evidence to support other measures such as physiological functions is available. OBJECTIVE: To investigate the effects of a usual (UP) versus a high protein (HP) diet on muscle function, cognitive function, quality of life and biochemical regulators of protein metabolism. DESIGN: A randomised intervention study was conducted with 23 healthy males (aged 19-31 yrs). All subjects consumed a Usual Protein (UP) diet (1.5 g protein/kg BW) for a 1-wk run-in period before the intervention period where they were assigned to either a UP or a High Protein (HP) diet (3.0 g protein/kg BW) for 3-wks with controlled intake of food and beverages. Blood and urine samples were taken along with measurements of physiological functions at baseline and at the end of the intervention period. RESULTS: The HP group improved their reaction time significantly compared with the UP group. Branched chain amino acids and phenylalanine in plasma were significantly increased following the HP diet, which may explain the improved reaction time. CONCLUSION: Healthy young males fed a HP diet improved reaction time. No adverse effects of the HP diet were observed. This trial was registered at www.clinicaltrials.gov as NCT00621231.