Abundant Plasma Proteins Research Articles

Feasibility of a plasma bioassay to assess oxidative protection of low-density lipoproteins by high-density lipoproteins

Traditionally, the impact of lipoproteins on vascular disease has been evaluated in light of their quantity, that is, cholesterol content, in plasma. However, recent studies of high-density lipoproteins (HDLs) have focused on functionality with regard to atheroprotection. For example, bioassays have emerged to assess the ability of HDL, in its near native plasma environment, to promote cholesterol removal (efflux) from cells. As a result, attention has focused on developing plasma-based assays for other putative HDL protective functions including protecting low-density lipoproteins (LDLs) from oxidative damage. To determine the feasibility of such an assay in a complex sample such as plasma, we evaluated the contribution of HDL vs other plasma factors in preventing LDL oxidation. We separated normolipidemic human plasma by gel filtration chromatography and assessed each fraction for its ability to prevent LDL modification by water soluble radical and copper-initiated oxidation mechanisms. Using proteomics and selective precipitation methods, we identified major antioxidative contributions for fibrinogen, immunoglobulin G, albumin, and small soluble molecules like uric acid and ascorbate, with albumin being especially dominant in copper-initiated mechanisms. HDL particles were minor contributors (∼1%-2%) to the antioxidant capacity of plasma, irrespective of oxidation mechanism. Given the overwhelming background of antioxidant capacity inherent to highly abundant plasma proteins, specific bioassays of HDL antioxidative function will likely require its complete separation from plasma.

Genetic Variation in 9p21 and the Plasma Proteome.

Single nucleotide polymorphisms (SNPs) in the noncoding region of 9p21 have been associated with cardiovascular disease (CVD), but the mechanisms by which these genetic variants contribute to the pathogenesis of CVD remain unknown since no annotated proteins are present in this region of DNA. The objective of the current study was to determine if 9p21 genotypes are associated with distinct plasma proteins in young adults. Subjects were 1611 young adults aged 20-29 years from the Toronto Nutrigenomics and Health Study (1098 females and 513 males). DNA was isolated from fasting blood to analyze four SNPs in 9p21 (rs2383206, rs10757274, rs10757278, and rs1333049). Abundant plasma proteins ( n = 54) were measured using liquid chromatography multiple reaction monitoring mass spectrometry. Analysis of covariance was used to determine differences in plasma proteins between genotypes. In Caucasians ( n = 524), four SNPs were associated with apolipoprotein E and two with haptoglobin-β-chain concentration. In East Asians ( n = 388), three SNPs were associated with α-1b-Glycoprotein, two with apolipoprotein B-100,one with apolipoprotein E and one withhaptoglobin-β-chain concentration. In South Asians ( n = 117), one SNP was associated with apolipoprotein B-100 concentration. Our findings suggest that 9p21 genotypes may play a role in various pathophysiological pathways that contribute to risk of CVD in early adulthood that might be distinct among different ethnocultural groups.

Anticalin® Proteins as Therapeutic Agents in Human Diseases.

Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central β-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life. Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Rα). Moreover, Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity. For example, PRS-343, which has recently entered clinical-stage development, combines an agonistic Anticalin targeting the costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 (HER2), thus offering a novel treatment option in immuno-oncology.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

O1.7. PROTEOMIC ANALYSIS OF BLOOD BASED SAMPLES FROM THE OPTiMiSE (OPTIMIZATION OF TREATMENT AND MANAGEMENT OF SCHIZOPHRENIA IN EUROPE) STUDY POINT TOWARDS COMPLEMENT PATHWAY PROTEIN CHANGES

BackgroundThe OPTiMiSE (Optimization of Treatment and Management of Schizophrenia in Europe) trial may help in the identification of predictors of treatment response. Medication naïve patients with first episode schizophrenia or schizophreniform disorder were enrolled in the study and treated open-label for a four-week period with amisulpride. PANSS ratings were undertaken at baseline and following the four-week treatment. 30 non-remitters (as defined by the Andreasen criteria) with the worst change in PANSS scores and the 30 remitters with the best change in PANSS scores were selected to represent good and poor outcome groups.MethodsWe compared proteomic markers in serum collected prior to treatment in 30 patients who subsequently showed a good response to amisulpride (“responders”, and 30 patients who did not show a good response (“non-responders”). Serum samples were depleted using High Performance Liquid Chromatography (HPLC) attached to a MARS column to remove the 14 most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin). The groups were matched for ethnicity, gender and age.50 µg from each sample were reduced, alkylated, tryptically digested, then zip-tipped to concentrate and purify. Samples were then run for a 90-min gradient on a Thermo Scientific Q- Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. Raw MS data were processed by MaxQuant software and searched against the human Uniprot database, for label free quantitation of peptides and proteins. False discovery rates (FDR) were set at 1% for both peptide and protein levels in target/decoy to minimize false positives. The match between runs feature was utilized.ResultsFour hundred and sixty-four protein identifications were obtained. Samples were excluded where >30% of proteins were missing, and we used imputation, where the missingness depends upon a threshold of detection. This left 228 proteins for analysis. Of these, 21 were significantly different between responders and non-responders (p<0.05), one was FDR positive, one at trend FDR level. Pathway analysis (KEGG, David NIH) of the significant proteins determined “complement and coagulation cascades” to be the top pathway affected with six proteins from the list assigned to the pathway. These were CFI, C4A, C6, F9, VWF and SERPING1, all found to be up-regulated and with p-values ranging from 0.002 to 0.044. C6 is a constituent of the membrane attack complex (MAC) that plays a key role in the innate and adaptive immune response, while CFI belongs to the alternative pathway, C4A belongs to the classical pathway and F9 and VWF play roles in the intrinsic coagulation pathway.DiscussionThese data complement results by Sekar et al., implicating excessive complement activity in the development of schizophrenia. Our data identifies the complement proteins in treatment response and this is also consistent with our previous findings of up-regulation of the complement pathway among those at risk of future psychotic experiences.

Neonatal Fc receptor is involved in the protection of fibrinogen after its intake in peripheral blood mononuclear cells

BackgroundFibrinogen is a central player in the blood coagulation cascade and one of the most abundant plasma proteins. This glycoprotein also triggers important events (e.g., cell spreading, the respiratory burst and degranulation) in neutrophil cells via a αMβ2 integrin-mediated binding to the cell surface. Yet, little is known about the interaction of fibrinogen with leukocytes other than neutrophils or stimulated monocytes, although high amounts of fibrinogen protein can also be found in lymphocytes, particularly in T-cells. The aim of the present work is to unveil the dynamics and the function of fibrinogen intake in T-cells.MethodsUsing the Jurkat cell line as a T-cells model we performed fibrinogen intake/competition experiments. Moreover, by means of a targeted gene knock-down by RNA-interference, we investigated the dynamics of the intake mechanism.ResultsHere we show that (i) fibrinogen, although not expressed in human peripheral blood mononuclear cells, can be internalized by these cells; (ii) fibrinogen internalization curves show a hyperbolic behavior, which is affected by the presence of serum in the medium, (iii) FITC-conjugated fibrinogen is released and re-internalized by adjacent cells, (iv) the presence of human serum albumin (HSA) or immunoglobulin G (IgG), which are both protected from intracellular degradation by the interaction with the neonatal Fc receptor (FcRn), results in a decreased amount of internalized fibrinogen, and (v) FcRn-knockdown affects the dynamics of fibrinogen internalization.ConclusionsWe demonstrated here for the first time that fibrinogen can be internalized and released by T-lymphocyte cells. Moreover, we showed that the presence of serum, HSA or IgG in the culture medium results in a reduction of the amount of internalized fibrinogen in these cells. Thus, we obtained experimental evidence for the expression of FcRn in T-lymphocyte cells and we propose this receptor as involved in the protection of fibrinogen from intracellular lysosomal degradation.

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD.We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln).With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense.Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.

Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma

BackgroundPlasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.MethodsBy using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science).ResultsOur data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit.ConclusionsWe demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

An update on medium- and low-abundant blood plasma proteome of horse

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation – time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

Plasma proteomics reveals coagulation, inflammation, and metabolic shifts in H-type hypertension patients with and without acute ischemic stroke.

Systematic profiling of a larger portion of circulating plasma proteome provide opportunities for unbiased discovery of novel markers to improve diagnostic, therapeutic, or predictive accuracy. This study aimed to identify differentially expressed proteins (DEPs) in plasma that could provide overall insight into the molecular changes of both H- type hypertension (HH) and HH-related acute ischemic stroke (AIS). This study used an iTRAQ-based LC-MS/MS proteomics approach to screen for plasma DEPs in HH patients with and without AIS, and controls. After excluding highly abundant plasma proteins, more than 600 proteins, and their relative levels, were identified. Of these, 26 DEPs, each showing > 1.2-fold change, were identified in HH and HH-related AIS patients compared with controls. Bioinformatics analysis revealed that these DEPs were enriched in 21 functional gene ontology items; “blood coagulation” was the most predominant pathway showing enrichment. Of these, eight DEPs were located in the hub position of networks involved with protein-protein interactions. AT-3, CRP, ApoB, and AHSG were further validated in each group by enzyme-linked immune sorbent assays. Comparing HH-related AIS with HH, the areas under the curve for AT-3, CRP, ApoB, and AHSG were 0.698, 0.892, 0.626, and 0.847, respectively. This proteomic profiling study provided enhanced pathophysiological understanding of the regulatory processes involved in coagulation, inflammation, and metabolism, and identified a panel of novel biomarkers for detecting HH-related AIS during its pre-stroke stage.

Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome.

Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

The rs10993994 functional polymorphism in the MSMB gene promoter increase the risk of prostate cancer in an Iranian population

Microseminoprotein-beta (MSMB) is one of the most abundant proteins in human seminal plasma. The current study was aimed to assess the impact of MSMB rs10993994, rs12770171, rs10994385 variants on prostate cancer (PCa) risk in a sample of Iranian population. This case-control study was executed on 169 pathologically confirmed PCa patients and 182 benign prostatic hyperplasia (BPH). Genotyping was performed by using the PCR-RFLP method. Our findings showed that rs10993994 variant significantly increased the risk of PCa in codominant (OR=1.64, 95%CI=1.04–2.59, P=0.038, CT vs TT) and overdominant (OR=1.64, 95%CI=1.03–2.43, P=0.039, TC vs TT+CC) models. However, no significant association was observed among rs10994385 G>C and rs12770171 C>T polymorphisms and PCa risk.In summary, the findings showed that MSMB rs10993994 polymorphism increased the risk of PCa in an Iranian population. To verify our findings, well-designed studies with larger sample sizes and representing various ethnicities are needed.

Increasing the accuracy of proteomic typing by decellularisation of amyloid tissue biopsies

Diagnosis and treatment of systemic amyloidosis depend on accurate identification of the specific amyloid fibril protein forming the tissue deposits. Confirmation of monoclonal immunoglobulin light chain amyloidosis (AL), requiring cytotoxic chemotherapy, and avoidance of such treatment in non-AL amyloidosis, are particularly important. Proteomic analysis characterises amyloid proteins directly. It complements immunohistochemical staining of amyloid to identify fibril proteins and gene sequencing to identify mutations in the fibril precursors. However, proteomics sometimes detects more than one potentially amyloidogenic protein, especially immunoglobulins and transthyretin which are abundant plasma proteins. Ambiguous results are most challenging in the elderly as both AL and transthyretin (ATTR) amyloidosis are usually present in this group. We have lately described a procedure for tissue decellularisation which retains the structure, integrity and composition of amyloid but removes proteins that are not integrated within the deposits. Here we show that use of this procedure before proteomic analysis eliminates ambiguity and improves diagnostic accuracy. SignificanceUnequivocal identification of the protein causing amyloidosis disease is crucial for correct diagnosis and treatment. As a proof of principle, we selected a number of cardiac and fat tissue biopsies from patients with various types of amyloidosis and show that a classical procedure of decellularisation enhances the specificity of the identification of the culprit protein reducing ambiguity and the risk of misdiagnosis.

Effective Interactions and Colloidal Stability of Bovine γ-Globulin in Solution.

Interactions and phase behavior of γ-globulins are of fundamental interest in biophysical and pharmaceutical research, as these are among the most abundant proteins in blood plasma. In this work, we report the characterization of the oligomeric state of bovine γ-globulin, the effective protein-protein interactions, and the colloidal stability in aqueous solution as a function of protein concentration and ionic strength. Classical biochemical techniques, such as size exclusion chromatography (SEC) and gel electrophoresis, together with small-angle X-ray and neutron scattering (SAXS/SANS), were employed for this study. The results show that bovine γ-globulin solutions are dominated by monomer and idiotype anti-idiotype dimer. Despite the flexibility and highly nonspherical shape of the protein, a simple model with a disk-type form factor and a structure factor of a square-well potential provide a satisfying description of the scattering data. The overall interactions are attractive and the strength decreases with increasing protein concentration, or adding buffer or salts. For higher protein volume fraction (>7%), the model would imply a strong particle-particle correlation which does not appear in the experimental data. This mismatch is most likely due to the smearing effect of the conformation change of proteins in solution. The stability of γ-globulin solutions is highly sensitive to protein concentration, ionic strength, and the type of added salts, such as NaCl, Na2SO4, and NaSCN. For solutions below 50 mg/mL and at low ionic strengths (<0.1 M), protein aggregation is most likely due to subpopulations of IgG molecules with attractive patches of complementary surface charge. This effect is reduced for higher protein concentration due to self-buffering effects. For high ionic strength (>1 M), typical salting-in (with NaSCN) and salting-out effects (with NaCl and Na2SO4) are observed. Results are further discussed in comparison with current studies in the literature on monoclonal antibodies.

Mass Spectrometry-Based Serum Proteomics for Biomarker Discovery and Validation.

Blood protein measurements are used frequently in the clinic in the assessment of patient health. Nevertheless, there remains the need for new biomarkers with better diagnostic specificities. With the advent of improved technology for bioanalysis and the growth of biobanks including collections from specific disease risk cohorts, the plasma proteome has remained a target of proteomics research toward the characterization of disease-related biomarkers. The following protocol presents a workflow for serum/plasma proteomics including details of sample preparation both with and without immunoaffinity depletion of the most abundant plasma proteins and methodology for selected reaction monitoring mass spectrometry validation.

In-Depth, Reproducible Analysis of Human Plasma Using IgY 14 and SuperMix Immunodepletion.

Identification of cancer and other disease biomarkers in human plasma has been exceptionally challenging due to the complex nature of plasma and the presence of a moderate number of high- and medium-abundance proteins which mask low-abundance proteins of interest. As a result, immunoaffinity depletion formats combining multiple antibodies to target the most abundant plasma proteins have become the first stage in most plasma proteome discovery schemes. This protocol describes the use of tandem IgY 14 and SuperMix immunoaffinity depletion to reproducibly remove >99% of total plasma protein. This greatly increases the depth of analysis of human plasma proteomes. Depleted plasma samples can then be analyzed in a single high-resolution LC-MS/MS run on a Q Exactive Plus mass spectrometer, followed by label-free quantitation. If greater depth of analysis is desired, the depleted plasma can be further fractionated by separating the sample for a short distance on a 1D SDS gel and cutting the gel into uniform slices prior to trypsin digestion. Alternatively, the depleted plasma can be reduced, alkylated, and digested with trypsin followed by high-pH reversed-phase HPLC separation.

Urinary Ceruloplasmin Concentration Predicts Development of Kidney Disease in Sickle Cell Disease Patients

BACKGROUND: Chronic kidney disease (CKD) is a prevalent complication of sickle cell disease (SCD) and associated with early mortality. Hemoglobinuria is a risk factor for the development and progression of CKD. Discovery and validation of non-invasive biomarkers for early stage renal disease are needed to facilitate optimal CKD treatment. Mass-spectrometry analysis of patient urine is a modern method for biomarker discovery. Urine from patients with late stages of glomerular disease contains a large number of abundant plasma proteins that overwhelm and complicate mass-spectrometry analysis. Thus, analysis of samples collected before the onset of kidney disease is a promising approach. Abnormal renal iron metabolism including cortical iron deposition is characteristic of SCD nephropathy. Ceruloplasmin is a ferrioxidase that is important facilitator of cellular iron export by ferroportin and of iron binding by transferrin.OBJECTIVES: We aimed to use mass-spectrometry to determine urinary biomarkers in SCD patients without renal disease that may predict the development of CKD and to validate the biomarkers by ELISA.METHODS: Mass-spectrometry analysis was performed on urine samples from eight University of Illinois at Chicago (UIC) SCD patients without CKD. Proteins were identified using Proteome Discoverer 1.4 and quantified using SIEVE 2.0 program. Ceruloplasmin concentrations were determined by ELISA in these eight subjects for the validation of our mass spectrometry analysis findings plus an additional 12 UIC SCD patients without CKD. Urine ceruloplasmin and free hemoglobin concentrations were determined by ELISA in an additional 34 UIC SCD patients with CKD stage ranging from 0-5.RESULTS: Label-free quantitative proteomic analysis of urine samples collected from SCD patients without CKD showed greater ceruloplasmin levels in 2 samples with hemoglobinuria versus 6 samples without hemoglboinuria (37.4-fold, p=2.7x10-8). Analysis of all twenty non-CKD samples by ELISA showed 2.5-fold higher levels of ceruloplasmin in hemoglobinuria samples (p=0.003). To determine whether urine ceruloplasmin correlated with CKD stage, we analyzed an additional 34 samples with and without CKD. Samples with CKD stages 2-5 (N=12) demonstrated higher levels of ceruloplasmin than stages 0-1 (N=22) (1.7-fold increase, p=0.008) (Figure 1A). In an additional analysis of these results, individual CKD disease stage correlated with urinary concentrations of ceruloplasmin (N=34, r=0.49, p=0.0035) (Figure 1B) and cell free hemoglobin (N=34, r=0.45, p=0.007) (Figure 1C). The correlation of ceruloplasmin concentration with CKD stage in the SCD patients showed high sensitivity and specificity (area under curve 90.7±6.8; p-value=0.018) by ROCK analysis. Finally, urinary ceruloplasmin concentration demonstrated a strong correlation with free hemoglobin concentration (r=0.79).CONCLUSIONS: Urinary ceruloplasmin may complement urinary free hemoglobin as a non-invasive biomarker of risk for CKD in SCD patients.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, 5G12MD007597 and K23HL125984. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of −16% and +37%, respectively (P < 10−13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten‐protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Biological significanceThough many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research.

A comparative analysis of serum albumin from different species to determine a natural source of albumin that might be useful for human therapy

Abstract Objectives Serum albumin (SA) is one of the most abundant proteins in human plasma and performs functions for protein transport, scavenges for free radicals, and regulates osmotic pressure. SA has been used for therapy in humans with several diseases including hypovolemia, hypoalbuminemia, and chronic liver disease. However, the most appropriate source of albumin for medical applications remains unclear, especially in the case of viral infections, which complicates finding viable donors. Substitution of human serum albumin (HSA) with albumin from other natural sources is a viable alternative. Therefore, we elucidated the similarity in character, structure and evolutionary relationship among serum albumin isolated from seven different species. Methods We compared the sequence, structure, and properties of SA from different species using an in-silico approach. Results These data suggested that SA has sequence polymorphism that clusters based on closely related-species. However, these polymorphisms do not change the three-dimensional structure of the protein; this may serve to maintain its function as a transporter. The Gallus gallus albumin has the lowest number of the epitopes that closely resemble HSA. Conclusion This study is crucial in providing explicit information about the structural similarity of albumin isolated from other species compared to HSA. The Gallus gallus SA might be used as a primary natural source of albumin where warranted for human therapy.