Analysis of membrane microdomain-associated proteins in the insular cortex of post-mortem human brain

Membrane microdomains (MM) are membrane rafts within the cell membrane enriched in cholesterol and glycosphingolipids that have been implicated in the trafficking and sorting of membrane proteins, secretory and endocytotic pathways, and signal transduction. To date, MM have not been characterised in the human brain. We reason that by identifying MM in the normal human cortex, we may better understand the molecular mechanisms of human brain dysfunction. To characterize the protein composition of MM in the human brain, we have carried out a comprehensive proteomic analysis of detergent resistant membranes (DRMs) associated proteins derived from human postmortem insular cortex using 1-DE separation prior to LC coupled to MS/MS or GeLC-MS/MS. Eighty five proteins were identified including 57 unique to human brain cortex DRMs (by comparison with DRM proteins reported in other cell types). High levels of signal transduction, cell adhesion, cell transport and cell trafficking proteins were identified including synaptic proteins such as synapsin II and synaptic vesicle membrane protein, mitochondrial proteins such as ATPase subunits and metabolic enzymes such as malate dehydrogenase. This data will facilitate our understanding of protein expression changes within membranes in candidate brain regions in human brain diseases such as schizophrenia, bipolar disorder and other psychiatric and neurodegenerative disorders.     

Analysis of protein profiling studies of β‐thalassemia/Hb E disease

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in β‐thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha‐1‐antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium‐binding protein, microtubule‐actin cross‐linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin‐2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified “response to inorganic substances” as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of β‐thalassemia/Hb E disease is starting to emerge.     

Hemoglobin depletion from red blood cell cytosol reveals new proteins in 2-D gel-based proteomics study

Proteomics studies to identify proteins in the erythrocyte cytosol have been largely affected by the huge abundance of hemoglobin (Hb), which masks the detection of other proteins in the 2-D gel-based separation. We have depleted Hb effectively from erythrocyte cytosol using cation exchange chromatography and have detected more than 600 protein spots in the Hb depleted hemolysate using 2-DE. We have so far identified 59 proteins in the Hb-depleted cytosol of normal erythrocytes, including 10 proteins not identified before.     

Differential regulation of redox proteins and chaperones in HbEβ-thalassemia erythrocyte proteome

Purpose: In (hemoglobin, Hb) HbEβ‐thalassemia, HbE (β‐26 Glu→Lys) interacts with β‐thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ‐thalassemic patients compared to normal. Experimental design: We have used 2‐DE and MALDI‐MS/MS‐based techniques to investigate the differential proteome profiling of membrane and Hb‐depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ‐thalassemia patients and normal volunteers. Results: Our study showed that redox regulators such as peroxiredoxin 2, Cu‐Zn superoxide dismutase and thioredoxin and chaperones such as α‐hemoglobin stabilizing protein and HSP‐70 were upregulated in HbEβ‐thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin‐based junctional complex in the membrane skeleton of HbEβ‐thalassemic erythrocytes upon detection of low molecular weight fragments of β‐spectrin and decrease in β‐actin and dematin content. Conclusion and clinical relevance: We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.     

Predicting protein subchloroplast locations: the 10th anniversary

Chloroplast is a type of subcellular organelle in green plants and algae. It is the main subcellular organelle for conducting photosynthetic process. The proteins, which localize within the chloroplast, are responsible for the photosynthetic process at molecular level. The chloroplast can be further divided into several compartments. Proteins in different compartments are related to different steps in the photosynthetic process. Since the molecular function of a protein is highly correlated to the exact cellular localization, pinpointing the subchloroplast location of a chloroplast protein is an important step towards the understanding of its role in the photosynthetic process. Experimental process for determining protein subchloroplast location is always costly and time consuming. Therefore, computational approaches were developed to predict the protein subchloroplast locations from the primary sequences. Over the last decades, more than a dozen studies have tried to predict protein subchloroplast locations with machine learning methods. Various sequence features and various machine learning algorithms have been introduced in this research topic. In this review, we collected the comprehensive information of all existing studies regarding the prediction of protein subchloroplast locations. We compare these studies in the aspects of benchmarking datasets, sequence features, machine learning algorithms, predictive performances, and the implementation availability. We summarized the progress and current status in this special research topic. We also try to figure out the most possible future works in predicting protein subchloroplast locations. We hope this review not only list all existing works, but also serve the readers as a useful resource for quickly grasping the big picture of this research topic.We also hope this review work can be a starting point of future methodology studies regarding the prediction of protein subchloroplast locations.     

Comparison of two-dimensional gel electrophoresis based and shotgun strategies in the study of plasma membrane proteome

Membrane proteins play important roles in various plasma membrane (PM) activities such as signal transduction and cell recognition. However, a comprehensive proteomic study of membrane proteins remains difficult. Different strategies have been employed to study PM proteome, but little effort has been made to systematically evaluate them. In the present work, liver PM was prepared by subcellular fractionation and an aliquot was washed by sodium carbonate. After evaluation of the PM fraction by electron microscopy and Western blotting, proteins in both original and carbonate washed PM were identified by either 2-DE coupled MALDI-TOF-MS or shotgun strategies. Then protein characteristics such as molecular weight, pI, grand average hydrophobicity, subcellular location, and transmembrane domains were systematically compared. The comparative analysis showed that shotgun strategies were more suitable to identify membrane proteins, while 2-DE-based strategies may serve as a complement. Furthermore, carbonate washing obviously enriched the integral membrane proteins. All the results suggested that the strategy combining carbonate washing and shotgun identification was the optimum strategy to study human liver PM proteome. Using this strategy, 260 high-confidence proteins were identified, wherein 139 were integral membrane proteins which had 1-17 transmembrane domains.     

Differential expression of red cell proteins in hemoglobinopathy

Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbEβ thalassemia.     

Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid

Purpose: Biomarkers are needed in cystic fibrosis (CF) to understand disease progression, assess response to therapy, and enrich enrollment for clinical trials. Aptamer‐based proteomics have proven useful in blood samples. The aim is to evaluate proteins in bronchoalveolar lavage fluid (BALF) in CF children compared to controls and identify endotypes during CF exacerbations. Experimental design: BALF is collected clinically from 50 patients with CF and nine disease controls, processed, and stored per protocol. BALF supernatants are analyzed for 1129 proteins by aptamer approach (SOMAscan proteomics platform). Proteins are compared across groups and used for pathway analysis. Endotypes are identified within the CF group. Results: CF BALF has increased concentrations of neutrophil elastase, myeloperoxidase, and decreased concentration of protein folding and host defense proteins. Pathways that distinguished CF subjects included interferon gamma signaling, membrane trafficking, and phospholipid metabolism. In the CF group, unbiased analysis of proteins identified two distinct endotypes that differed based on BALF white blood cell and neutrophil counts and detection of CF pathogens. Conclusions and clinical relevance: Proteomic analysis of the CF airway demonstrates a complex environment of proteins and pathways. This work provides evidence that aptamer‐based proteomics can differentiate between groups and can determine endotypes within CF.     

Mapping the 5-50-kDa fraction of human amniotic fluid proteins by 2-DE and ESI-MS

This report presents a proteomic analysis and provides a reference map of the 5-50-kDa components of normal amniotic fluid collected in gestational weeks 16-18. Early amniocentesis samples were pooled and proteins with molecular mass lower than albumin were separated by gel filtration chromatography. The 2-DE protocol was optimized for the separation of the small proteins and peptides in the fraction of interest. A total of 132 Coomassie blue-stained protein spots were analyzed, following in-gel tryptic digestion, by ESI-MS/MS and 49 different gene products were identified. The treatment with alkaline phosphatase caused the shift of the phosphoisoforms of insulin-like growth factor-binding protein-1 and of the N-terminal osteopontin fragment. Of the 33 full-length proteins identified in the 2-DE profile, 23 had not been previously detected in the amniotic fluid and, of these, 22 are not present in the human plasma proteome under physiological conditions. Fragments of 16 larger proteins were identified and the sequence coverage data revealed that several correspond to autonomous domains that may have biological roles on their own. Several of the detected proteins and peptides appear to be involved in critical regulatory processes associated with placentation and early development, thus representing potential markers of various physiological or pathological conditions.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Shotgun MS proteomic analysis of bronchoalveolar lavage fluid in normal subjects

We provide a review of proteomic techniques used to characterize the bronchoalveolar lavage fluid (BALF) proteome of normal healthy subjects. Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. The proteomic techniques used to study normal BALF include protein separation by 2DE, whereby proteins were identified by comparison to a reference gel as well as high pressure liquid chromatography (HPLC)-MS/MS, also known as shotgun proteomics. We summarize recent progress using shotgun MS technologies to define the normal BALF proteome. Surprisingly, we find that despite advances in shotgun proteomic technologies over the course of the last 10 years, which have resulted in greater numbers of proteins being identified, the functional landscape of normal BALF proteome was similarly described by all methods examined.     

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.     

Proteome analysis of a plasma membrane-enriched fraction at the placental feto-maternal barrier

Purpose : We want to identify proteins that are part of or associated with the plasma membrane of the human feto‐maternal barrier, which is crucially important for nutrient, gas, and waste exchange between the mother and the fetus. All transfer processes occur through one specialized endothelial cell layer, the multinuclear syncytiotrophoblast (STB). Specifically, the apical plasma membrane of the STB interacts with the maternal blood and is the site of initial transport processes across the placenta. Experimental design : We used a proteomic approach that employed the enrichment of apical STB membranes isolated from healthy placentae by ultracentrifugation and saccharose gradient centrifugation steps in combination with 1‐D SDS‐PAGE and ESI‐MS analysis. Results : We identified 296 different proteins, 175 of which were integral and peripheral membrane proteins, partially containing 1–12 transmembrane domains or lipid anchors. One hundred and sixty‐one proteins (54%) were allocated to the plasma membrane. Conclusions and clinical relevance : A high number of transporters, receptors, and proteins involved in signal transduction processes and vesicular trafficking were identified for the first time at the feto‐maternal barrier. Our results are valuable sources for further studies of the cell physiology of the healthy placenta at the time of birth or the pathophysiology of several pregnancy disorders.     

利用奇异值分解最小二乘法(SVDLS)解析蛋白质的现场光谱电化学圆二色(CD)谱

圆二色谱仪是研究蛋白质二级结构的有力工具，能在远紫外区测出蛋白质的二级结构的各种构象。通常的蛋白质二级结构有α-螺旋、β-折叠、反β-折叠、β-转角及无规则卷曲五种构象，实际观测到的CD谱是这几种二级结构构象CD谱的线性加和。本文利用了奇异值分解最小二乘算法(SVDLS)，并编译相关程序解析现场外加电压电解条件下的蛋白质溶液的CD谱，得到几种二级结构构象的组分分布。所得结果与文献报道上的其他方法如SELCON3等相比较，证明是一种快速和有效的方法。     

Deep depletion of abundant serum proteins reveals low-abundant proteins as potential biomarkers for human ovarian cancer

Epithelial ovarian cancer (EOC) ranks fifth as a cause of cancer deaths in women. Current diagnostic and monitoring markers have limited reliability for the detection of disease. We have tested the possibility of identifying candidate biomarkers present at low nanogram to picogram levels after removing both the 12 most abundant and 77 moderately abundant proteins from serum samples of EOC patients using antibody affinity columns. We showed that this approach allows the identification of proteins that are expressed at nanogram per liter levels in the serum. Using ICAT/MS/MS analysis, we identified 51 proteins that are differentially expressed by at least twofold. These proteins include leucine-rich α-2-glycoprotein, matrix metalloproteinase-9 (MMP-9), inter-α-trypsin inhibitor heavy chain H1, insulin-like growth factor-binding protein 6, insulin-like growth factor-binding protein 3, isoform 1 of epidermal growth factor receptor, angiopoietin-like protein 3 (ANGPTL3) and phosphatidylcholine-sterol acyltransferase. We confirmed the differential expression of MMP9 and ANGPTL3 in normal and ovarian cancer sera by ELISA assays. Further robust clinical evaluation of the candidate markers identified is necessary.     

Toward the Proteome of the Human Peripheral Blood Eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom^® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10^?3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.     

The murine plasma protein response to polymicrobial intra-abdominal sepsis

Protein biomarkers in the peripheral blood could potentially be used as early indicators of sepsis and a means to stratify patients for clinical trials. Although individual molecular markers have been proposed for sepsis, none has clinical utility. The global changes in plasma proteins over the clinical course of sepsis have not been characterized using proteomic methods. We used cecal ligation and puncture to induce polymicrobial sepsis in mice and generated plasma protein profiles using 2‐D DIGE of plasma from septic mice and surgical controls. Replicate cohorts (n = 3) of 4–7 animals each were used to identify 62 gel features that changed significantly (Student's t‐test, p<0.05). We identified a suite of plasma proteins that describe uniquely the host plasma response to polymicrobial septic insult. Principal components analysis of protein abundance showed that ～90% of the variability between samples was due to sepsis. In addition to canonical acute phase proteins, we identified proteins that are associated with metabolic changes (e.g. α‐2 HS glycoprotein and zinc α‐2 glycoprotein) consistent with the pathophysiology of sepsis. The panel of sepsis‐associated molecular markers identified herein may prove useful in the diagnosis and categorization of sepsis.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

2-D DIGE and MALDI-TOF-MS analysis of the serum proteome in human osteosarcoma

We performed 2-D DIGE on proteins prepared from serum obtained from patients with osteosarcoma (OS) and controls, to identify differentially expressed proteins that might serve as serum biomarkers for OS prognosis. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. We compared serum samples from four individuals with OS to four age- and sex-matched healthy controls. We identified 24 protein spot-features that were significantly increased, and 34 that were significantly decreased in serum from patients with OS relative to the controls. The MS analysis revealed 18 unique proteins that were increased, and 25 unique proteins that were decreased in OS serum samples. Western blot and ELISA analysis confirmed increased levels of amyloid-related serum protein (SAA) in the OS serum samples. The increased expression levels of SAA were decreased after using MTX and cisplatin combination chemotherapy, and were further decreased after operation. Moreover, increased expression levels of sera SAA were seen in the relapsed patients. Our results suggested that the determination of serum SAA in OS patients might be utilized as a marker for relapse and in evaluation of the efficacy of therapy.