Proteomic Analysis Of Human Serum Research Articles

Quantitative proteomic analysis of human serum using tandem mass tags to predict cardiovascular risks in patients with psoriasis

Although biomarker candidates associated with psoriasis have been suggested, those for predicting the risk of cardiovascular disease (CVD) early in patients with psoriasis are lacking. We aimed to identify candidate biomarkers that can predict the occurrence of CVD in psoriasis patients. We pursued quantitative proteomic analysis of serum samples composed of three groups: psoriasis patients with and those without CVD risk factors, and healthy controls. Age/Sex-matched serum samples were selected and labeled with 16-plex tandem mass tag (TMT) and analyzed using liquid chromatography-mass spectrometry and subsequent verification with ELISA. Of the 184 proteins that showed statistical significance (P-value < 0.05) among the three groups according to TMT-based quantitative analysis, 98 proteins showed significant differences (> 2.0-fold) between the psoriasis groups with and without CVD risk factors. Verification by ELISA revealed that caldesmon (CALD1), myeloid cell nuclear differentiation antigen (MNDA), and zyxin (ZYX) levels were significantly increased in the psoriasis group with CVD risk factors. Further network analysis identified pathways including integrin signaling, which could be related to platelet aggregation, and actin cytoskeleton signaling. Three novel candidates (MNDA, ZYX, and CALD1) could be potential biomarkers for predicting CVD risks in psoriasis patients. We expect these biomarker candidates can be used to predict CVD risk in psoriasis patients in clinical settings although further studies including large validation are needed.

Proteomic Analysis of Human Serum for Patients at Different Pathological Stages of Hepatic Fibrosis.

Background Hepatic fibrosis is a severe liver disease that has threatened human health for a long time. In order to undergo timely and adequate therapy, it is important for patients to obtain an accurate diagnosis of fibrosis. Laboratory inspection methods have been efficient in distinguishing between advanced hepatic fibrosis stages (F3, F4), but the identification of early stages of fibrosis has not been achieved. The development of proteomics may provide us with a new direction to identify the stages of fibrosis. Methods We established serum proteomic maps for patients with hepatic fibrosis at different stages and identified differential expression of proteins between fibrosis stages through ultra-high-performance liquid chromatography tandem mass spectrometry proteomic analysis. Results From the proteomic profiles of the serum of patients with different stages of liver fibrosis, a total of 1,338 proteins were identified. Among three early fibrosis stages (control, F1, and F2), 55 differential proteins were identified, but no proteins simultaneously exhibited differential expression between control, F1, and F2. Differential proteins were detected in the comparison between different fibrosis stages. Significant differences were found between advanced fibrosis stages (F2-vs.-F3 and F3-vs.-F4) through a series of statistical analysis, including hierarchical clustering, Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction network analysis. The differential proteins identified by GO annotation were associated with biological processes (mainly platelet degranulation and cell adhesion), molecular functions, and cellular components. Conclusions All potential biomarkers identified between the stages of fibrosis could be key points in determining the fibrosis staging. The differences between early stages may provide a useful reference in addressing the challenge of early fibrosis staging.

Proteomic Analysis of Human Serum from Patients with Chronic Kidney Disease

Chronic kidney disease (CKD) is an important public health problem in the world. The aim of our research was to identify novel potential serum biomarkers of renal injury. ELISA assay showed that cytokines and chemokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, FGFb, G-CSF, GM-CSF, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-1bb, RANTES, TNF-α and VEGF were significantly higher (R > 0.6, p value < 0.05) in the serum of patients with CKD compared to healthy subjects, and they were positively correlated with well-established markers (urea and creatinine). The multiple reaction monitoring (MRM) quantification method revealed that levels of HSP90B2, AAT, IGSF22, CUL5, PKCE, APOA4, APOE, APOA1, CCDC171, CCDC43, VIL1, Antigen KI-67, NKRF, APPBP2, CAPRI and most complement system proteins were increased in serum of CKD patients compared to the healthy group. Among complement system proteins, the C8G subunit was significantly decreased three-fold in patients with CKD. However, only AAT and HSP90B2 were positively correlated with well-established markers and, therefore, could be proposed as potential biomarkers for CKD.

Comparative proteomic analysis of human serum before and after liver transplantation using quantitative proteomics

Liver cancer is the second leading cause of cancer mortality worldwide. Safer and more effective diagnostic methods for liver cancer are desirable, and biomarkers represent a potentially alternative method for diagnosis. The present study was designed to identify liver cancer biomarkers. We quantified the changes in serum protein levels between liver transplantation and healthy (control) females using isobaric tags for relative and absolute quantitation (iTRAQ) as well as proteomic analysis. A total of 1399 proteins were identified; of these, three proteins showed significantly different concentrations between the before transplantation group and the control group. These proteins may thus be relevant to liver cancer and constitute potential liver cancer biomarkers.

Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.

Proteomic analysis of human serum of osteopenic women after dried plum treatment

Dried plum has the ability to prevent and reverse bone loss as evidenced by an increase in bone mineral density as well as changes in bone turnover biomarkers such as insulin growth factor‐1, bone‐specific alkaline phosphatase, tartrate‐resistant acid phosphatase‐5b and sclerostin. However, it is not known whether dried plum affects other proteins in human serum that may influence bone turnover. The objective of this study was to explore if dried plum affects proteins in human serum other than the known bone turnover biomarkers related in the reversal of bone loss in osteopenic postmenopausal women using isobaric tags for relative and absolute quantification (iTRAQ). Serum samples were obtained from forty‐eight osteopenic women (65–79 years old) who were randomly assigned to one of three treatment groups for six months: 1) control 2) 100g dried plum or 3) 50g dried plum. Samples were collected at baseline and after three and six months of intervention. The iTRAQ quantitative proteomics approach was conducted to explore the proteome affected by dried plum treatment. iTRAQ results identified 51 unique proteins that were upregulated with at least a 2‐fold difference in change between control and treatment groups (p&lt;0.05). Twenty‐four proteins were significantly upregulated only at 3‐months, five proteins at both 3‐ and 6‐months and eighteen at 6‐months only. Of the relevant proteins, ten proteins relate to inflammation, at least 5 relate to immunological function in general, five relate to lipid metabolism and one has a known direct association with bone metabolism. Dried plum treatment resulted in an upregulation of 51 unique proteins in human serum, which may or may not be linked to bone turnover. Future studies must be conducted to determine if these are in fact related to bone turnover processes.Support or Funding InformationPilot Award from UCSD CTRI (UL1 TR000100)

Proteomic analysis of human serum from diabetic retinopathy.

To establish and compare serum proteomic of diabetic retinopathy (DR) patients in various phases and discuss pathogenesis of DR so as to find out possible serum specific molecular markers for early diagnosis of DR. Thirty-two subjects were divided into four groups: one group of eight type 2 diabetes mellitus (T2DM) patients without apparent DR (No-DR, NDR), one group of eight T2DM patients with non-proliferative diabetic retinopathy (NPDR), one group of eight T2DM patients with proliferative diabetic retinopathy (PDR) and one group of eight healthy volunteer participants. Two dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to establish differential protein expression profiles in four groups. Matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF-TOF MS) was applied to identify mass spectrometry of differential proteins and analyze follow-up bioinformatics. 2D-DIGE maps of serum protein were satisfactory obtained from NDR, NPDR, PDR and normal control groups. Twenty-six different proteins spots were screened (the volume ratio was >1.5 based on DeCyder software analysis). Twenty-four of them were verified and two of them were not. Fifteen proteins were verified. Most of them were high-abundant proteins in serum. The four relatively low-abundant ones were beta 2-glycoprotein I (β(2)-GPI), alpha2-HS-glycoprotein(AHSG), alpha1-acid glycoprotein(α(1)-AGP) and apolipoprotein A-1(apo A-1). β(2)-GPI expression was gradually increased in the development of DR but unrelated to the severity of DR. The volume ratio of β(2)-GPI is 1.54, 2.43, and 2.84 in NDR, NPDR and PDR group respectively compared with normal control group. Serum proteomic analysis of 2D-DIGE combined with MALDI-TOF-TOF MS is feasible to be applied in the study of DR. β(2)-GPI probably takes part in the process of DR occurrence and development and it could be a candidate biomarker on DR diagnosis in early phase.

Preliminary proteomic analysis of human serum from patients with laryngeal carcinoma

To better evaluate the serum protein alterations in patients with laryngeal carcinoma during surgical treatment process, a comparative proteomic analysis of human serum from patients with laryngeal carcinoma between pre- and post-operation group was performed using two-dimensional gel electrophoresis, in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and database searching. Statistical analysis indicated that 16 gel spots corresponding to 12 proteins altered their expression significantly between the two groups of patients with laryngeal carcinoma. Of these 12 proteins, 6 proteins were up-regulated in the pre-operation group. The subsequent Western blot confirmed the results of proteome analysis. Measurement of MDA and SOD levels in serum combining with bioinformatics analysis indicated the potential roles of the oxidant stress and immune response as target in monitoring and treating laryngeal carcinoma. The study provides new insight into the laryngeal carcinoma development and treatment, and the identified proteins are warranted to be further studied.

Proteomic analysis of human serum for Finding pathogenic factors and potential biomarkers in preeclampsia

Objective(s)To apply a novel proteomic method to discover potential pathogenic factors and biomarkers of preeclampsia. Study designSera from five patients complicated with preeclampsia and five healthy pregnant controls were separately pooled. Each pool was treated with peptide ligand library beads (PLLBs) to remove high abundance proteins by affinity and thus enrich low abundance proteins. The proteins from the eluate were analyzed by a combination of 1D Gel-LC-MS/MS. Protein expression levels were quantified using spectral counts and the extracted ion current. Results1172 unique proteins in preeclampsia and 1149 in healthy controls were identified in the present study. 51 proteins were differentially expressed between preeclampsia and healthy pregnant women including chorionic somatommammptropin hormone (CSH) and fibulin-1. 31 proteins identified were up-regulated and 20 were down-regulated. ConclusionsThe results demonstrate that peptide ligand library combining with 1D gel-LC-MS/MS analysis is an efficient method to identify differentially expressed proteins in sera and two biological processes of complement and coagulation activations and lipid metabolism were involved in the pathogenesis of preeclampsia.

Comparison of 1‐D and 2‐D LC MS/MS methods for proteomic analysis of human serum

1-D and 2-D LC methods were utilized for proteome analysis of undepleted human serum. Separation of peptides in 2-D LC was performed either with strong cation exchange (SCX)-RP chromatography or with an RP-RP 2-D LC approach. Peptides were identified by MS/MS using a data-independent acquisition approach. A peptide retention prediction model was used to highlight the potential false-positive peptide identifications. When applying selected data filtration, we identified 52 proteins based on 316 peptides in serum in 1-D LC setup. One hundred and eighty-four proteins/1036 peptides and 142 proteins/905 peptides were identified in RP-RP and SCX-RP 2-D LC, respectively. The performance of both 2-D LC methods for proteomic analysis is critically compared.

Proteomic analysis of human serum from patients with temporal lobe epilepsy

=Abstract= Purpose: Epilepsy affects more than 0.5% of the world's population. It has a large genetic component and is caused by electrical hyperexcitability in the central nervous system. Despite its prevalence, the disease lacks definitive diagnostic serological biomarkers. To identify potential biomarkers for epilepsy by a convenient method, we analyzed the expression of serum proteins, reflecting alterations in the patient's proteomes. Methods: We compared two-dimensional electrophoretic band patterns of human sera from eight patients with temporal lobe epilepsy (TLE) with those of eight control subjects. The differentially expressed bands were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. Results: Twelve proteins were differentially expressed in the TLE group, of which 6 were identified. Expression of haptoglobin Hp2, PRO2675, immunoglobulin heavy chain constant region gamma 2, an unnamed protein, and three unidentified proteins were upregulated in serum from the patients with TLE, whereas those of major histocompatibility complex (MHC) class I antigen, plasma retinol-binding protein precursor, and three unidentified proteins were downregulated in these patients. After resection of the epileptogenic zone, the expressions of MHC class I antigen, immunoglobulin heavy chain constant region gamma 2, two of the downregulated unidentified proteins, and one of the upregulated unidentified proteins returned to the normal range. Conclusion: The 12 serum proteins in this study are potentially useful biomarkers for the diagnosis and monitoring of TLE. (Korean J Pediatr 2009;52:567-575)

Proteomic study of serum using gel chromatography and MALDI-TOF MS reveals diagnostic biomarkers in male patients with liver-cancer

Human serum has been widely employed clinically for diagnosing various fatal diseases. However, the concentration of most proteins in human serum is too low to be directly measured using normal analytical methods. In order to obtain reliable analytical results from proteomic analysis of human serum, appropriate sample preparation is essential. A combined off-line analytical technique of gel chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been successfully established to separate proteins for MS analysis. Using these combined techniques, 176 mass signal peaks of proteins/peptides were found in 6 of 18 fractions from normal male serum (NMS) in the presence of buffer consisting of NH 4HCO 3 and acetonitrile. A simple gel chromatography column packed with Sephadex G-50 removed most signal-suppressing compounds such as salts and high abundance proteins (HAP). The molecular mass to charge ( m/z) ratios of differential peptides revealed in serum of male patient with liver-cancer (LCMPS) compared to NMS were 5365, 5644 and 6462, and these peptides can be used as biomarkers to clinically diagnose liver-cancer. The simple and convenient chromatographic method described here is not only superior to recently described HPLC separation for MS analysis, but also reveals many novel and significant serum biomarkers for the clinical diagnosis of various diseases.

Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.

Proteomic Analysis of Human Serum by Two-Dimensional Differential Gel Electrophoresis after Depletion of High-Abundant Proteins

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.