Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)–embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT–embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis. (J Histochem Cytochem 57:849–860, 2009)     

Effect of purified recombinant human erythropoietin on anemia in rats with experimental renal failure induced by five-sixth nephrectomy

Recombinant human erythropoietin (rHuEPO) was purified from the conditioned media of Chinese hamster ovary cells with a transfected human erythropoietin gene. We investigated the effects of the rHuEPO in rats with renal anemia induced by partial nephrectomy. Five-sixth nephrectomy resulted in renal failure with anemia. Twenty-five days after the operation plasma urea nitrogen was increased about 2.5 times, and the red blood cell count, hematocrit, and hemoglobin concentration fell to 85% of normal. The reticulocyte count and plasma erythropoietin level did not change such as they do in patients with anemia due to chronic renal failure. Both total red blood cell volume and the plasma iron turnover rate were depressed in five-sixth nephrectomized rats compared with normal rats.The five-sixth nephrectomized rats were injected with rHuEPO (60 IU/kg) intravenously every second day for a total of six injections. After three injections of rHuEPO, circulation volume of total red blood cells was increased from 9.9 ml to 14.6 ml, and the plasma iron turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day, and the reticulocyte count was also increased. After six injections, a marked increase of the red blood cell count, hematocrit, and hemoglobin concentration were observed. Plasma urea nitrogen and the creatinine levels as indications for renal function did not change after rHuEPO administration in both normal and five-sixth nephrectomized rats.In conclusion rHuEPO has a potent erythropoietic action and it is possible to cure the anemia caused by renal failure.     

Overexpression of sorcin in multidrug resistant human leukemia cells and its role in regulating cell apoptosis

In an attempt to identify novel proteins involved in the emergence of multidrug resistance (MDR) in leukemia cells, we adopted a proteomics approach to analyze protein expression patterns in leukemia cell lines, K562, and its MDR counterpart, K562/A02. Combining high-resolution two-dimensional gel electrophoresis and mass spectrometry, we compared the protein expression profiles between K562 and K562/A02. A total number of 22 protein spots with altered abundances of more than 2-fold were detected and 14 proteins were successfully identified. Consistent with our previous observations by cDNA microarray, sorcin, a 22-kDa calcium-binding protein, was also identified by this proteomic approach with a 10.4-fold up-regulation in K562/A02 cells. Overexpression of sorcin protein in K562 cells by gene transfection led to significantly reduced cytosolic calcium level and increased resistance to cell apoptosis. Further, leukemia cell lines over-expressing sorcin also showed up-regulation of Bcl-2, along with decreased level of Bax. Taken together, our results suggest that sorcin plays an important role in the emergence of MDR in leukemia cells via regulating cell apoptosis pathways, thus may represent both a new MDR marker for prognosis and a good target for anti-MDR drug development.     

Identification of pathways associated with invasive behavior by ovarian cancer cells using multidimensional protein identification technology (MudPIT)

Proteomic profiling has emerged as a useful tool for identifying tissue alterations in disease states including malignant transformation. The aim of this study was to reveal expression profiles associated with the highly motile/invasive ovarian cancer cell phenotype. Six ovarian cancer cell lines were subjected to proteomic characterization using multidimensional protein identification technology (MudPIT), and evaluated for their motile/invasive behavior, so that these parameters could be compared. Within whole cell extracts of the ovarian cancer cells, MudPIT identified proteins that mapped to 2245 unique genes. Western blot analysis for selected proteins confirmed the expression profiles revealed by MudPIT, demonstrating the fidelity of this high-throughput analysis. Unsupervised cluster analysis partitioned the cell lines in a manner that reflected their motile/invasive capacity. A comparison of protein expression profiles between cell lines of high (group 1) versus low (group 2) motile/invasive capacity revealed 300 proteins that were differentially expressed, of which 196 proteins were significantly upregulated in group 1. Protein network and KEGG pathway analysis indicated a functional interplay between proteins up-regulated in group 1 cells, with increased expression of several key members of the actin cytoskeleton, extracellular matrix (ECM) and focal adhesion pathways. These proteomic expression profiles can be utilized to distinguish highly motile, aggressive ovarian cancer cells from lesser invasive ones, and could prove to be essential in the development of more effective strategies that target pivotal cell signaling pathways used by cancer cells during local invasion and distant metastasis.     

CSF proteomic analysis reveals persistent iron deficiency-induced alterations in non-human primate infants

Iron deficiency (ID) anemia during infancy results in long-term neurological consequences, yet the mediating mechanisms remain unclear. Infant monkeys often become naturally anemic during the first 6 months of life, presenting an opportunity to determine the effect of developmental iron deficiency. After weaning, animals were chosen randomly for supplementation with oral iron or, fed a standard commercial chow diet. The control group was never iron deficient. ID anemia was corrected by 12 months in both groups, as indicated by hematological parameters. CSF was collected for proteomic analysis at 12 months of age to assess the impact of developmental ID on the brain. The CSF proteome for both formerly iron deficient groups was similar and revealed 12 proteins with expression levels altered at least twofold. These proteins were identified by matrix assisted laser desorption ionization time-of-flight spectrometry and included prostaglandin D synthase, olfactory receptors and glial fibrillary acidic protein. Thus the proteomic analysis reveals a persistent effect of ID and provides insights into reports of disturbed sleep, hypomyelination and other behavioral alterations associated with ID. Furthermore, alterations in the CSF proteome despite normal hematologic parameters indicate that there is a hierarchical system that prioritizes repletion of red cell mass at the expense of the brain.     

Protein Expression Profiles Characterize Distinct Features of Mouse Cerebral Cortices at Different Developmental Stages

The proper development of the mammalian cerebral cortex requires precise protein synthesis and accurate regulation of protein expression levels. To reveal signatures of protein expression in developing mouse cortices, we here generate proteomic profiles of cortices at embryonic and postnatal stages using tandem mass spectrometry (MS/MS). We found that protein expression profiles are mostly consistent with biological features of the developing cortex. Gene Ontology (GO) and KEGG pathway analyses demonstrate conserved molecules that maintain cortical development such as proteins involved in metabolism. GO and KEGG pathway analyses further identify differentially expressed proteins that function at specific stages, for example proteins regulating the cell cycle in the embryonic cortex, and proteins controlling axon guidance in the postnatal cortex, suggesting that distinct protein expression profiles determine biological events in the developing cortex. Furthermore, the STRING network analysis has revealed that many proteins control a single biological event, such as the cell cycle regulation, through cohesive interactions, indicating a complex network regulation in the cortex. Our study has identified protein networks that control the cortical development and has provided a protein reference for further investigation of protein interactions in the cortex.     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

A variant glucose-6-phosphate dehydrogenase Gd(-) Chiapas associated with moderate enzyme deficiency and occasional hemolytic anemia

Summary Erythrocyte glucose-6-phosphate deficiency is an X-chromosomal-linked hereditary trait often associated with hemolytic anemia. This report defines a new variant designated as Gd(-) Chiapas, which was found in a subject with occasional hemolytic jaundice. The red cell enzyme activity of the subject is about 15% of normal. The variant enzyme is thermolabile in vitro and has faster-than-normal anodal electrophoretic mobility and stronger-than-normal substrate affinity. The patient's hemolytic problem might be correlated with instability of the variant enzyme under physiologic stress.This study was supported in part by the US Public Health Service Grant HL-15125.     

ER/PR阳性和阴性乳腺癌的定量蛋白质组学和生物信息学比较研究

目的:建立雌/孕激素受体(ER/PR)阴性和阳性乳腺癌的蛋白质表达谱,寻找ER/PR阴性和阳性乳腺癌中差异表达蛋白,为乳腺癌患者提供新的预后预测指标和治疗新靶点。方法:应用蛋白质组学i TRAQ技术建立ER/PR阳性和阴性乳腺癌的蛋白质差异表达谱,鉴定两组乳腺癌的差异表达蛋白,对部分差异表达蛋白进行生物信息学分析,包括蛋白功能注释和分类GO分析和KEGG通路分析。结果:应用i TRAQ蛋白质组学技术对乳腺癌组织进行了蛋白组学分析,鉴定出ER/PR阳性和阴性组间有差异表达的蛋白4999种,以ER/PR阳性:ER/PR阴性≥3为上调标准,确定ER/PR阳性组上调蛋白101种。以ER/PR阳性:ER/PR阴性≤0.5为下调标准,ER/PR阳性组下调蛋白122种。GO分析结果显示ER/PR受体阴性和阳性乳腺癌的差异表达蛋白的分子功能、生物过程、细胞定位较为复杂,并且在上调蛋白和下调蛋白上存在分布差异。KEGG通路分析发现部分差异表达蛋白涉及201条信号通路。结论:ER/PR阳性和阴性乳腺癌间存在差异表达蛋白,这些蛋白涉及复杂的分子功能、生物过程和信号通路。     

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.     

In-Depth,Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature

Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.     

Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye

To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238 protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC.     

不同分化程度的鼻咽癌细胞系质膜差异蛋白质组分析

本研究以CNE1和CNE2为材料,采用亚细胞蛋白质组研究方法研究不同分化程度鼻咽癌细胞系的差异蛋白质．首先用Percoll密度梯度离心法获得高纯度质膜,通过双向凝胶电泳分离、PDQuest软件分析后找出在肿瘤细胞中表达变化的蛋白质点,再用基质辅助激光解析电离飞行时间串联质谱（MALDI-TOF/TOF-MS）进行鉴定,共鉴定到9个具有2倍或2倍以上差异的蛋白质.这些表达差异的蛋白质参与了细胞分化、代谢及细胞信号传导过程．我们对其中5个蛋白质进行了实时定量PCR分析,对其中4个蛋白质的表达进行了免疫印迹验证．本试验为研究不同分化程度的鼻咽癌提供了一种蛋白质组研究方法,并且找到了galectin-1、annexin Ⅱ等一些可能与分化相关的蛋白质.这些数据对于研究鼻咽癌的生物学特性具有非常重要的意义．     

Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency

The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity.     

Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.     

Risk Factors of Pulmonary Hypertension in Brazilian Patients with Sickle Cell Anemia

This study was a prospective cross-sectional cohort study of 125 patients with sickle cell anemia (SS) between the ages of 16 to 60 years. Enrolled patients were followed-up prospectively for 15 months. Demographic, clinical, hematological and routine biochemical data were obtained on all patients. Six-minute walk test and Doppler Echocardiography were performed on all patients. A tricuspid regurgitant jet velocity (TRJV) < 2.5 m/sec was considered normal, 2.5 ≤ TRJV ≤ 3.0 was considered mild-moderate and > 3.0 m/sec, severe. Patients with abnormal TRJV were significantly older and more anemic, had significantly higher lactate dehydrogenase (LDH) levels, reticulocyte count and incidence of death. The logistic multimodal model implemented for the 125 patients indicated that age was the covariate that influenced the outcome of normal or abnormal TRJV with a cutoff age of thirty-two years. The survival rate for the group of patients with creatinine (Cr) > 1.0 mg/dL was lower than the group with Cr ≤ 1 and normal TRJV. A coefficient matrix showed that the LDH values were weakly correlated with the reticulocyte count but strongly correlated with hemoglobin suggesting that the TRJV values were not correlated with the hemolytic rate but with anemia. Ten patients died during the follow-up of whom 7 had TRJV > 2.5 m/sec. Acute chest syndrome was the most common cause of death followed by sepsis. In conclusion, this study shows that patients with SS older than thirty-two years with high LDH, elevated TRJV, severe anemia and Cr > 1 have poor prognosis and may be at risk of having pulmonary hypertension and should undergo RHC.