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.     

A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening

Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via 2-D DIGE that were expressed at either higher or lower levels (>three-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the ER stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.     

Proteomic analysis in cerebrospinal fluid of patients with atypical nonketotic hyperglycinemia and pulmonary hypertension - A pilot study

A variant phenotype of nonketotic hyperglycinemia has been described by our group associated with pulmonary hypertension. The aim of this study is to investigate the cerebrospinal fluid proteomes to get an insight into this neurodegenerative process producing leukoencephalopathy with white matter spongiform degeneration. DIGE and MALDI-TOF-TOF analyses were performed to carry out the proteomic study of four patients against three normal controls and one additional control of a classical nonketotic hyperglycinemia. The differential proteomic analysis showed a displacement of some series of spots toward the acidic side. The shifted proteins showed a high degree of carbonylation and increased methionine sulfoxidation was found in cystatin C and in vitamin-D-binding protein. These findings in addition to the increase of serum malondialdehyde concentration provide evidence of an oxidative stress in the patients under study, which is probably systemic rather than mainly confined to the CNS. The similarities of our findings with those found in other neurodegenerative diseases suggest that oxidative damage is commonly involved in these pathologies. DIGE technology improves the 2-D PAGE differential analysis and it is suitable in proteomic studies with a small number of cases.     

Anatomic site‐specific proteomic signatures of gastrointestinal stromal tumors

The gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. Its clinical course ranges widely from a curable disorder to a highly malignant disease. Although its clinical and molecular characteristics depend on the anatomic site of origin, the molecular background of GIST arising in different anatomical site has not been studied yet. To investigate the proteomic background of GIST, we examined the proteomic features corresponding to the anatomic site of tumor origin. Comparison of the proteomic profile of gastric (23 cases) and small intestinal (9 cases) GIST by 2‐DE revealed 105 protein spots with significantly different intensity (p <0.01) between the two groups. Mass spectrometric study identified 68 distinct proteins for these 105 protein spots, including cancer‐associated ones such as prohibitin, pigment epithelium‐derived factor, and alpha‐actinin 4. The intensity of 37/105 (35.2%) protein spots was significantly concordant with the corresponding mRNA levels (p <0.01). Although both 2‐D DIGE and microarray experiments showed significant up‐regulation of vimentin expression in small intestinal GIST, Western blotting did not show a significant difference between the two groups. In conclusion, our study demonstrates the proteins specially expressed in GIST depending on their site of origin, as well as the unique advantage offered by use of proteomics to acquire such data. The identified proteins may provide clues to understanding the different characteristics of GIST depending on their site of origin.     

Colorectal cancer proteomics, molecular characterization and biomarker discovery

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression.     

Considerations for powering a clinical proteomics study: Normal variability in the human plasma proteome

Proteomics is increasingly being applied to the human plasma proteome to identify biomarkers of disease for use in non‐invasive assays. 2‐D DIGE, simultaneously analysing thousands of protein spots quantitatively and maintaining protein isoform information, is one technique adopted. Sufficient numbers of samples must be analysed to achieve statistical power; however, few reported studies have analysed inherent variability in the plasma proteome by 2‐D DIGE to allow power calculations. This study analysed plasma from 60 healthy volunteers by 2‐D DIGE. Two samples were taken, 7 days apart, allowing estimation of sensitivity of detection of differences in spot intensity between two groups using either a longitudinal (paired) or non‐paired design. Parameters for differences were: two‐fold normalised volume change, α of 0.05 and power of 0.8. Using groups of 20 samples, alterations in 1742 spots could be detected with longitudinal sampling, and in 1206 between non‐paired groups. Interbatch gel variability was small relative to the detection parameters, indicating robustness and reproducibility of 2‐D DIGE for analysing large sample sets. In summary, 20 samples can allow detection of a large number of proteomic alterations by 2‐D DIGE in human plasma, the sensitivity of detecting differences was greatly improved by longitudinal sampling and the technology was robust across batches.     

The glomerular proteome in a model of chronic kidney disease

Adequate kidney function is crucial in sustaining vertebrate homeostasis. Certain diseases can diminish renal function and lead to end-stage renal disease. Diabetes mellitus and hypertension are the main causes of glomerulosclerosis and albuminuria in adults. The molecular mechanisms that trigger these maladaptive changes are still unsatisfyingly described. We previously introduced 2-D DIGE in combination with focused tissue isolation methods to analyze protein expression in glomeruli. Glomeruli, the crucial compartments in albuminuric renal diseases, were extracted using magnetic particles from subtotally nephrectomized FVB mice (n?=?6); this 5/6 nephrectomy in FVB mice is a model of chronic kidney disease. Analysis of protein expression levels from glomerular protein lysates was performed using 2-D DIGE and compared with glomerular protein lysates from mice that underwent sham surgery. The comparison of about 2100 detectable spots between both groups revealed 48 protein spots that showed significant differential expression. Of those, 33 proteins could be identified using nanoLC-ESI MS. The metalloproteinase meprin 1 alpha, the beta galactoside-binding-lectin galectin-1 and dimethylarginine dimethylaminohydrolase 1, a key enzyme in NO metabolism, were found to be differentially regulated, thus implying a role in the pathogenesis and pathophysiology of progressive kidney disease. In conclusion, 2-D DIGE protein analysis of smallest sample sizes from specific organ compartments provides focused protein expression results, which help in gaining an understanding of the molecular mechanisms of chronic kidney disease.     

HCV inhibits antigen processing and presentation and induces oxidative stress response in gastric mucosa

Productive hepatitis C virus (HCV) infection appears to be primarily confined to the liver. However, a wide variety of extrahepatic disease manifestations are associated with the infection and HCV RNA has been frequently detected in gastric mucosa. The present study aims to determine molecular alterations present in vivo in the stomach where HCV expression does not induce a carcinoma but a lymphoma, thus extending the knowledge of alterations in intracellular pathways consequent to HCV infection. We compared, by 2-D DIGE, the gastric protein expression profile from six HCV positive and six HCV negative samples lacking neoplastic or dysplastic conditions. In HCV positive tissue we observed a down regulation of proteins involved in MHC maturation and assembly, antigen processing and presentation and ER stress, in addition to an up regulation of proteins involved in cellular oxidative stress responses. Ubiquinol-cytochrome-C-reductase (UQCRFS1), part of the mitochondrial respiratory chain complex-III, was identified as the most up regulated protein. Data were confirmed by Western blot and immunohistochemistry. Our results demonstrate a HCV negative influence on the different pathways that determine antigen processing and presentation via MHC-I and the cellular attempts to counteract HCV induced oxidative stress. Both these processes facilitate immune escape and cell survival and probably contribute to HCV chronicization.     

Urine collected from diapers can be used for 2-D PAGE in infants and young children

Urinary proteomic profiling has potential to identify candidate biomarkers of renal injury in infants provided an adequate urine sample can be obtained. Although diapers are used to obtain urine for clinical evaluation, their use for proteomic analysis has not been investigated. We therefore performed feasibility studies on the use of diaper-extracted urine for 2-D PAGE. Pediatric waste urine (2–20 mL) was applied to gel-containing, non-gel and cotton-gauze diapers and then mechanically expressed. Urine volume and total protein were measured pre- and post-extraction. Proteins were separated via 2-D PAGE following application of urine (20–40 mL) to each matrix. 2-D PAGE was also performed on clinical specimens collected using each diaper type. Differences in the adsorption and retention of urine volume and protein were noted between matrices. Non-gel and cotton-gauze diapers provided the best protein/volume recovery and the lowest interference with the Bradford assay. 2-D PAGE was also successfully completed using urine samples from both cotton fiber matrices. Conversely, samples from low-gel diapers demonstrated poor protein separation and reproducibility. Diapers containing cotton–fiber matrices appear adequate for 2-D PAGE. Qualitative and quantitative analyses of resolved proteins using replicate, high-resolution gels will be required, however, before diaper-extracted urine can be applied in proteomic profiling.     

Proteomic expression analysis and comparison of protein and mRNA expression profiles in human malignant gliomas

Gliomas are highly heterogeneous and therapy resistant tumors with a poor prognosis. Novel experimental therapeutic approaches have shown some promising results, but often target specific molecular mechanisms or antigens, and careful characterization of the molecular subgroup of the tumors will therefore likely be important. Thorough investigations of gene and protein alterations are also important to better understand the tumorigenic mechanisms. We have undertaken a proteomic approach, using 2-D DIGE and LC-MS/MS protein identification, to investigate 38 human gliomas and normal brains. We show that the proteome profile can discriminate between normal brain and tumors, and between tumors of varying grade by a supervised classifier. Furthermore, an analysis of the identified proteins shows an enrichment of proteins associated to pathways known to be central in gliomas, such as MEK/Erk signaling and actin cytoskeleton. It also shows a shift between different glial fibrillary acidic protein (GFAP) representatives in different grades. In a previous study the gene expression profile was characterized in an almost identical set of tumors, which enabled a paired analysis of the gene and protein expression profiles. We show that there is often a weak correlation between the mRNA and protein level. This, together with the ability of proteomics to identify PTMs, emphasizes the benefit of characterization on a protein level.     

An optimum method designed for 2-D DIGE analysis of human arterial intima and media layers isolated by laser microdissection

The formation and progression of atherosclerotic lesions involve complex mechanisms which are still not fully understood. A variety of cell types from the distinct arterial layers are implicated in the whole process from lipid accumulation within the vascular wall to plaque development and final rupture. In the present work, we employ the combination of laser microdissection and pressure catapulting and 2-D DIGE saturation labeling to investigate the human intima and media sub-proteomes isolated from atherosclerotic (coronary and aorta) or non-atherosclerotic vessels (preatherosclerotic coronary arteries). Laser microdissection and pressure catapulting allows the specific isolation of regions of interest. In turn, DIGE saturation labeling overcomes the limitation of extensive microdissection times to recover the protein amount required to perform comparative 2-DE, particularly when dealing with tissue regions rich in myofilament proteins, which result in low protein recovery. The compatibility and optimum performance of both techniques were investigated in detail, paying special attention to tissue staining and protein solubilization. Since scarce amount of protein obtained from microdissected tissue made it impossible to directly perform protein identification from 2-DE spots by MS, we performed in-solution digestion followed by LC-MS/MS analysis of total protein extracts from intima and media in order to get an overall picture of protein composition. Proteins so identified confirm the nature of the isolated regions. Finally, similar spot resolution on 2-D DIGE gels was obtained for the different human artery types (coronary, aorta) and studied layers (intima, media), setting the basis for future clinical comparative studies.     

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.     

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.     

Proteomics and the lung: Analysis of bronchoalveolar lavage fluid

Our knowledge of the complex bronchoalveolar lavage fluid (BALF) proteome has increased significantly over the last decade; but still, there remain many aspects of the BALF proteome that need characterization. Current proteomic methodologies resolve proteins within limited dynamic ranges: thereby, being limited in their ability to examine important areas of the BALF proteome, such as low molecular weight, low abundance proteins. To ensure proper coverage of these proteins in the BALF proteome, a refined 2-DE standard operation protocol is presented, highlighting important issues in sample collection, sample preparation, and 2-D DIGE analysis. It is hoped that this will help advance the field of BALF proteomics, BALFomics, which has lagged behind similar biofluids such as plasma and serum.     

Identification of cancer-associated proteins by proteomics and downregulation of β-tropomyosin expression in colorectal adenoma and cancer

Elucidating the molecular mechanism underlying the development of adenoma, the major precursor lesion of colorectal cancer (CRC), would provide a basis for early detection, prevention as well as treatment of CRC. Using the highly sensitive 2-D DIGE method coupled with MS, we identified 24 differentially expressed proteins in adenoma tissues compared with matched normal colonic mucosa and CRC tissues. Fifteen proteins were downregulated and three proteins were upregulated in adenoma tissues when compared with individual-matched normal colonic mucosa. Five proteins were downregulated, while one protein was upregulated in adenoma tissues when compared with matched CRC tissues. A protein, β-tropomyosin (TM-β), recently suggested to be a biomarker of esophageal squamous carcinoma, was downregulated in both adenoma and CRC tissues. Additionally, the reduction in the level of TM-β in adenoma and CRC tissues was further validated by Western blotting (p<0.05) and RT-PCR (p<0.001). Our findings suggest that downregulation of TM-β is involved in the early development of CRC and that differentially expressed proteins might serve as potential biomarkers for detection of CRC.     

Peroxiredoxin 2 as a chemotherapy responsiveness biomarker candidate in osteosarcoma revealed by proteomics

Purpose : We aimed to identify novel chemotherapy responsiveness biomarkers for osteosarcoma (OS) by investigating the global protein expression profile of 12 biopsy samples from OS patients. Experimental design : Six patients were classified as good responders and six as poor responders, according to the Huvos grading system. The protein expression profiles obtained by 2‐D DIGE consisted of 2250 protein spots. Results : Among them, we identified 55 protein spots whose intensity was significantly different (Bonferroni adjusted p‐value<0.01) between the two patient groups. Mass spectrometric protein identification demonstrated that the 55 spots corresponded to 38 distinct gene products including peroxiredoxin 2 (PRDX 2). Use of a specific antibody against PRDX 2 confirmed the differential expression of PRDX 2 between good and poor responders, while PRDX 2 levels as measured by Western blotting correlated highly with their corresponding 2‐D DIGE values. The predictive value of PRDX 2 expression was further confirmed by examining an additional four OS cases using Western blotting. Conclusions and clinical relevance : These results establish PRDX 2 as a candidate for chemotherapy responsiveness marker in OS. Measuring PRDX 2 in biopsy samples before treatment may contribute to more effective management of OS.     

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14⁺ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.     

Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue.     

Vascular proteomics

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1α, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.     

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.