Comparative proteomic analysis of plasma of children with congenital heart disease

Congenital heart disease is one of the largest class of birth defects. Eight subjects with ventricular septal defect (VSD, a kind of congenital heart disease) and 11 health children were enrolled in tandem mass tags label‐based quantitative proteomic analysis to compare plasma proteins differentially abundance. A total of 66 proteins were significantly upregulated or downregulated in VSD patients compared with healthy children. These proteins were involved in pathways linked to platelet activation, fructose and mannose metabolism, complement and coagulation cascades, glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and carbon metabolism. The amount of ten proteins changed significantly (p < 0.05) in newly recruited 30 VSD compared with 15 control children, which were validated by ELISA. The areas under the receiver operating characteristic curve values of fructose‐bisphosphate aldolase B (ALDOB) and thymosin beta‐4 (Tβ4) were higher than those of other candidate proteins. ALDOB and Tβ4 might be potential biomarkers applied for identifying VSD in the further works.     

Metabolic Profiling Using Reversed Phase High Performance Liquid Chromatography: Analysis of Urine from Patients with Rheumatoid Arthritis

Abstract

Optimal conditions are described for the reversed phase HPLC separation of UV absorbing constituents including peptides and proteins in urine. Metabolic profiles have been obtained for urinary constituents in the molecular weight range > 1000, 1000–10,000, and > 10,000 for healthy controls, patients with rheumatoid arthritis and osteoarthritis. A computer program was employed to compare individual and groups of profiles. Considerable variation was encountered in the patterns of constituents and comparison of the metabolic profiles revealed no diagnostically significant differences between the various groups of subjects.     

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.     

Early diagnosis of lung cancer based on proteome analysis of exhaled breath condensate

A comparative study of the exhaled-breath-condensate (EBC) proteome that was obtained for four donor groups was carried out using ion cyclotron resonance mass spectrometry with electrospray ionization. The groups included subjects with diagnosed lung cancer, chronic obstructive pulmonary disease, community-acquired pneumonia, and healthy nonsmoking control subjects. More than 300 proteins were identified, while 19 of them were found in the EBC samples of the donors who were diagnosed with lung cancer in the early stages and are potentially significant in the development of a diagnostic lung-cancer biomarker panel. It was shown that the EBC protein profiles of different donor groups can be distinguished. It may be possible to highlight a specific protein group that is typical for certain conditions/diseases of the respiratory system. Thus, the EBC analysis could be a promising non-invasive method for early diagnosis of lung cancer.     

Serum protein profiling in patients with inflammatory bowel diseases using selective solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemometric data analysis

The identification of new biomarkers or a disease-related protein fingerprint for inflammatory bowel diseases (IBDs) represents a major task in the diagnosis, prognosis and pharmacological therapy. To address these issues, a simple and rapid analytical proteomic method for serum protein profiling based on selective beads-based solid-phase bulk extraction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and chemometric data analysis was developed. Serum proteins from healthy subjects (22) and patients with Crohn's disease (15) and ulcerative colitis (26) were selectively extracted according to reversed-phase (C18), strong anion-exchange (SAX) and metal ion affinity (IDA-Cu(II)) principles. This approach allowed enrichment of serum proteins/peptides due to the high interaction surface between analytes and the solid phase and high recovery due to the elution step performed directly on the MALDI-target plate. The MALDI-TOF MS serum protein profiles were acquired and, after a data pre-processing step, analyzed by linear discriminant analysis (LDA), a chemometric classification technique, in order to classify serum samples among healthy subjects and patients with inflammatory bowel diseases (IBDs). Since the high number of variables in the MALDI spectra (more than 16000 m/z values) prevents the use of LDA, the variables were reduced to 10-20 by features selection, thus allowing the evaluation of a pattern of m/z values with high discriminant power. Serum protein profiles obtained by reversed-phase extraction and the selection of 20 m/z values gave the best overall prediction ability (96.9%). The recognition of these m/z values may allow the identification of protein biomarkers involved in IBDs.     

A comparative protein profiling of lymphocytes from blood of patients with chronic lymphoid leukemia by high-resolution mass-spectrometry in search of new markers for heterogeneity and disease prognosis

To find new markers of chronic lymphoid leukemia heterogeneity, a comparison of the proteomic profiles of pathologic and healthy lymphocytes from blood was carried out by high-resolution mass spectrometry and label-free quantitative analysis. A list of the most typical proteins for the two groups was obtained. The differences in the proteomic profiles within the pathologic group, which were consistent with the heterogeneity of clinical background, were found. Two subsets within the area of the pathologic cells were formed using statistical analysis; the list of characteristic proteins for each subset was obtained.     

Proteomics and Detection of Uromodulin in First-time Renal Calculi Patients and Recurrent Renal Calculi Patients

Renal calculi disease or known as kidney stone disease is the most common urological disorder in both men and women, although it is more prevalent in men. The lifetime chance for an individual to develop renal calculi is ~10% whereas the risk of recurrence in a 10-year period is 74%. Therefore, a diagnostic tool for screening or detecting renal calculi is greatly needed. In this study, we analyze urinary protein profiles from patients with renal calculi for the first time (RC), healthy subjects (HS), and patients with recurrent renal calculi (RRC) to identify a biomarker for detecting the disease. Urinary proteins were isolated by salt precipitation and the proteins resolved by SDS-PAGE. Target proteins were analyzed with LC/MS/MS. Thirty-two proteins were identified from healthy subjects and patients. Uromodulin was the most abundant urinary protein in HS but was a very faint band if detected at all from those that formed renal calculi for the first time (p < 0.05). Yet the excreted levels of urinary uromodulin in RRC were similar to those of the HS suggesting that uromodulin is a reliable biomarker for only RC. In addition, a few immunoglobulins that were commonly found in the urine of both RC and RRC, which include Ig alpha heavy chain 1, Ig gamma-2 c region, Ig gamma-3 heavy chain disease protein, Ig heavy chain variable region, Ig heavy constant region gamma 4, and Ig heavy chain. Ig heavy chain Fab frag and antibody a5b7 chain B may serve as potential biomarkers for renal calculi disease.     

Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.     

Evaluation of biological monitoring markers using genomic and proteomic analysis for automobile emission inspectors and waste incinerating workers exposed to polycyclic aromatic hydrocarbons or 2,3,7,8,-tetracholrodedibenzo-p-dioxins

In this study, we investigated the effects of PAHs and dioxin on mRNA and plasma protein expression using genomic and proteomic analysis for automobile emission inspectors and waste incineration workers. About 54 workers from automobile emission inspection offices, 31 workers from waste incinerating company and 84 unexposed healthy subjects were enrolled in this study. Urine and air samples were collected and analyzed by HPLC and GC/MS. Comet assays were carried out to evaluate any DNA damage in mononuclear and polynuclear cells. A significant difference in Olive tail moments in mononuclear cells was observed between exposed and control subjects (P < 0.0001). To examine the differences of the gene expression profile in automobile emission inspectors and waste incineration workers, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 total genes. The gene expression profiles showed that 11 genes were up-regulated and 4 genes were down-regulated in waste incinerating workers as compared with controls. Plasma proteins were analyzed by 2-dimentional electrophoresis with pH 3-10 NL IPG Dry strip. The protein expression profiles showed that 8 proteins were up- regulated and 1 protein, haptoglobin, was down- regulated in automobile emission inspectors and waste incineration workers. Serum paraoxonase/ arylesterase was found only in the plasma of waste incineration workers. The expression of genes and proteins involved in oxidative stress were up-regulated in both automobile emission inspectors and waste incineration workers. Several proteins, such as transthyrethin, sarcolectin and haptoglobin, that were highly up- or down-regulated, could serve as biological monitoring markers for future study.     

Urinary protein profiling by liquid chromatography/tandem mass spectrometry: ADAM28 is overexpressed in bladder transitional cell carcinoma

Bladder cancer is the most common urological cancer with higher incidence rate in the endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to utilize the proteomic approach to establish urinary protein patterns of bladder cancer. The experimental results showed that most patients with bladder cancer had proteinuria or albuminuria. The urine arsenic concentrations of bladder cancer patients in BFD areas were significantly higher than those patients from non-BFD areas. In the proteomic analysis, the urinary proteome was identified by nano-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (nano-HPLC/ESI-MS/MS) followed by peptide fragmentation pattern analysis. We categorized 2782 unique proteins of which 89 proteins were identified with at least three unique matching peptide sequences. Among these 89 proteins, thirteen of them were not found in the control group and may represent proteins specific for bladder cancer. In this study, three proteins, SPINK5, ADAM28 and PTP1, were also confirmed by Western blotting and showed significant differential expression compared with the control group. ADAM28 may be used as a possible biomarker of bladder cancer.     

Metabonomic study of biochemical changes in urinary of type 2 diabetes mellitus patients after the treatment of sulfonylurea antidiabetic drugs based on ultra‐performance liquid chromatography/mass spectrometry

A metabonomic study on biochemical changes in the urine of type 2 diabetes mellitus (T2DM) patients after the treatment of sulfonylurea (SU) antidiabetic drugs was performed. An ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS) method was used to generate metabolic fingerprints for the metabonomic analysis of urinary samples obtained from 20 T2DM patients without any drug treatment and 20 T2DM patients treated with SU antidiabetic drugs and 20 normal glucose tolerance subjects. The resulting data were subjected to chemometric analysis (principal component analysis and partial least squares discriminant analysis) to investigate the effect of SU antidiabetic drugs on urinary metabolite profiles of T2DM patients. Biomarkers such as xanthine, phenylalanine, tryptophan, hippurate, phenylacetylglutamine, carnitine C8:1, carnitine C10:3, uric acid and citrate were found to be responsible for the separation of T2DM and SU‐treated groups, which indicates a potential effect of SU on energy metabolism, Tricarboxylic acid (TCA) cycle, gut microflora metabolism and oxidative stress. The study may be helpful to the understanding of the action of mechanism of SU antidiabetic drugs. Copyright © 2014 John Wiley & Sons, Ltd.     

Two-dimensional analysis of tear protein patterns of diabetic patients.

In diabetic patients, dry eye and other ocular surface diseases occur more often than in healthy subjects. The aim of this study was to analyze the tear protein patterns of patients suffering from diabetes mellitus type II (dia) and to compare them to the patterns of healthy volunteers (ctrl). Tear proteins of nonstimulated tears of 20 patients (ctrl n=10, dia n=10) were separated using two-dimensional electrophoretic techniques. The protein patterns of each group were analyzed by a multivariate analysis of discriminance. Furthermore, for all spots of each gel, a 50 x 50 variables pH/Mr (molecular weight) array was generated and subsequently analyzed by a multivariate analysis of discriminance. Additionally, an artificial neural network was trained using the matrix data as input and a sensitivity analysis was performed to figure out, which spots were the most important to differentiate between the tear protein patterns. In both groups a complex staining pattern could be obtained. In diabetic patients significantly more spots were detected compared to the control group (P<0.02). The analysis of discriminance found a highly significant difference between dia and ctrl (P<0.00001). Using the matrix data, the analysis of discriminance showed a significant difference between the two groups, too (P<0.0001). The sensitivity analysis by means of the artificial neural network revealed several spots that were more expressed or more frequently present in the diabetic group. Our findings reveal that the composition of tear proteins of diabetic patients is different from that of healthy subjects. The use of the two-dimensional electrophoretic technique could give more insight into the diabetic-related changes in the tear film composition.     

Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress

Two spring wheat varieties Ningchun 4 and Chinese Spring with good and poor resistance to abiotic stress, respectively, were used to investigate proteomic changes in the developing grains under drought stress by a comparative proteomics approach. A total of 152 protein spots showed at least twofold differences in abundance on two-dimensional electrophoresis (2-DE) maps, of which 28 and 68 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry, respectively. Of the 96 identified protein spots, six different expression patterns were found and they were involved in stress/defense/detoxification, carbohydrate metabolism, photosynthesis, nitrogen metabolism, storage proteins and some other important functions. Comparative proteomic analysis revealed that under the drought conditions the decreased degree of ascorbate peroxidases was more significant in Chinese Spring than in Ningchun 4 during grain development whereas translationally controlled tumor protein, which was significantly upregulated at 14 DAF, was present in Ningchun 4 and absent in Chinese Spring. The Rubisco large subunit displayed an upregulated expression pattern in Ningchun 4. In addition, two drought-tolerant proteins, triosephosphate isomerase and oxygen-evolving complex showed B and F type expression patterns in Chinese Spring, but D and B types in Ningchun 4, respectively. These differentially expressed proteins might be responsible for the stronger drought resistance of Ningchun 4 compared to Chinese Spring.     

Proteomics Profiling of the Urine of Patients with Hyperthyroidism after Anti-Thyroid Treatment

Hyperthyroidism, which is characterized by increased circulating thyroid hormone levels, alters the body’s metabolic and systemic hemodynamic balance and directly influences renal function. In this study, the urinary proteome of patients with hyperthyroidism was characterized using an untargeted proteomic approach with network analysis. Urine samples were collected from nine age-matched patients before and after carbimazole treatment. Differences in the abundance of urinary proteins between hyperthyroid and euthyroid states were determined using a 2D-DIGE coupled to MALDI-TOF mass spectrometry. Alterations in the abundance of urinary proteins, analyzed via Progenesis software, revealed a statistically significant difference in abundance in a total of 40 spots corresponding to 32 proteins, 25 up and 7 down (≥1.5-fold change, ANOVA, p ≤ 0.05). The proteins identified in the study are known to regulate processes associated with cellular metabolism, transport, and acute phase response. The notable upregulated urinary proteins were serotransferrin, transthyretin, serum albumin, ceruloplasmin, alpha-1B-glycoprotein, syntenin-1, and glutaminyl peptide cyclotransferase, whereas the three notable downregulated proteins were plasma kallikrein, protein glutamine gamma-glutamyl transferase, and serpin B3 (SERPINB3). Bioinformatic analysis using ingenuity pathway analysis (IPA) identified the dysregulation of pathways associated with cellular compromise, inflammatory response, cellular assembly, and organization and identified the involvement of the APP and AKT signaling pathways via their interactions with interleukins as the central nodes.     

非小细胞肺癌患者尿中Exosomes蛋白质组的差异表达分析

除血浆之外,尿液是另一种寻找潜在生物标志物的重要生物材料。本研究以200000×g超速离心法分离正常人和非小细胞肺癌(NSCLC)患者尿液中的Exosomes,运用1DSDS-PAGE对Exosomes蛋白质组进行分组,从电泳胶上切取正常组和疾病组的20～31kDa条带,胰蛋白酶酶解后,进行HPLC-CHIP-MS/MS分析,并通过UniProtKB/SWISS-PORT数据库搜索鉴定了24种蛋白质,其中在NSCLC患者尿液Exosomes蛋白质组中发现了8种差异表达蛋白,包括免疫球蛋白κ的3个片段、2种Ras相关蛋白、谷胱甘肽S转移酶A2、血清淀粉样P成分前体和磷脂酰乙醇胺结合蛋白1。     

A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

Background

A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.

Results

One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.

Conclusions

In B. mori larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in B. mori. A first attempt to that end allowed isolation of a B. mori LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

Effects of Hexane on Protein Profile,Solubility and Foaming Properties of Defatted Proteins Extracted from Tenebrio molitor Larvae

Inclusion of edible insects in human diets is increasingly promoted as a sustainable source of proteins with high nutritional value. While consumer acceptability remains the main challenge to their integration into Western food culture, the use of edible insects as meal and protein concentrate could decrease neophobia. The defatting of edible insects, mostly done with hexane, is the first step in producing protein ingredients. However, its impact on protein profiles and techno-functionality is still unclear. Consequently, this study compares the protein profiles of hexane-defatted and non-hexane-defatted yellow mealworm (Tenebrio molitor) meals and protein extracts, and evaluates the impact of hexane on protein solubility and foaming properties. Results showed that profiles for major proteins were similar between hexane-defatted and non-defatted samples, however some specific content differences (e.g., hexamerin 2) were observed and characterized using proteomic tools. Protein solubility was markedly lower for T. molitor meals compared to protein extracts. A large increase in the foaming capacity was observed for defatted fractions, whereas foam stability decreased similarly in all fractions. Consequently, although the hexane-defatting step was largely studied to produce edible insect protein ingredients, it is necessary to precisely understand its impact on their techno-functional properties for the development of food formulations.     

Complex phylogenetic profiling reveals fundamental genotype-phenotype associations

We have developed a computational technique refereed to as complex phylogenetic profiling. Our approach combines logic analyses of gene phylogenetic profiles and phenotype data. Logic analysis of phylogenetic profiles identifies sets of proteins whose presence or absence follows certain logic relationships. Our approach identifies phenotype specific logic, i.e. it identifies sets of proteins simultaneously present or absent only in genomes with a given phenotype. For example, for most genomes expressing phenotype A, the presence of protein C presumes the presence of protein B, while for other genomes (not expressing phenotype A) the presence of protein C presumes the absence of protein B. Application of complex phylogenetic profiling to bacterial data and several well studied phenotypes reveals genotype-phenotype associations on the level of fundamental biochemical pathways.