Apolipoprotein E knockout (apoE−/−) mouse is one of the most popular models for cardiovascular research, especially in the study of atherosclerosis. Naturally, large amount of studies try to uncover the role of apoE in atherosclerosis, and indeed apoE plays an important role in this pathogenesis. Kidney is an organ that contains lots of capillaries and also largely expresses apoE. Moreover, a protective role of apoE in kidney as an autocrine regulator has been demonstrated previously, however, the underlying mechanism is largely unknown.
In this study, comparative proteomics is for the first time used to identify the differential proteins in kidneys of apoE−/− and wild type (WT) mice, respectively, and we try to reveal the signaling network of apoE in mice kidney using bioinformatics analysis.
Our findings show that approximately 80 proteins are significantly differentially expressed in kidneys of apoE−/- and WT mice, and the signaling network correlated to apoE is successfully established by employing bioinformatics assay.
Taken together, we originally identify the proteins with differential expression and propose an apoE correlated molecular network in mice kidney. These findings further provide evidence of the role of apoE in mice kidney and a brand new perspective in the protection and treatment of kidney disease.
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Effectively identifying the proteins present in this cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum.
Two stromal cell lines were incubated with tetraacetylated sugar-azide analogs for 48 hours in serum free and low serum conditions. Sugar-azide labeled glycoproteins were covalently linked to alkyne-beads, followed by on-bead trypsin digestion and tandem mass spectrometry. The resulting glycoproteins were compared between media conditions, cell lines, and azide-sugar labels.
Alkyne-bead capture of sugar-azide modified glycoproteins in stromal cell culture media significantly improved the detection of lower abundance secreted glycoproteins compared to standard serum-free secretome preparations. Over 100 secreted glycoproteins were detected in each stromal cell line and significantly enriched relative to a standard secretome preparation.
Sugar-azide metabolic labeling is an effective way to enrich for secreted glycoproteins present in cell line secretomes, even in culture media supplemented with serum. The method has utility for identifying secreted stromal proteins associated with cancer progression and the epithelial-to-mesenchymal transition.
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Aortic aneurysm is a deceptively indolent disease that can cause severe complications such as aortic rupture and dissection. In the normal aorta, vascular smooth muscle cells within the medial layer produce and sustain the extracellular matrix that provides structural support but also retains soluble growth factors and regulates their distribution. Although the extracellular matrix is an obvious target to identify molecular processes leading to structural failure within the vessel wall, an in-depth proteomics analysis of this important sub-proteome has not been performed. Most proteomics analyses of the vasculature to date used homogenized tissue devoid of spatial information. In such homogenates, quantitative proteomics comparisons are hampered by the heterogeneity of clinical samples (i.e. cellular composition) and the dynamic range limitations stemming from highly abundant cellular proteins. An unbiased proteomics discovery approach targeting the extracellular matrix instead of the cellular proteome may decipher the complex, multivalent signals that are presented to cells during aortic remodelling. A better understanding of the extracellular matrix in healthy and diseased vessels will provide important pathogenic insights and has potential to reveal novel biomarkers.
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Cardiovascular disease (CVD) is the leading cause of death and loss of productive life years in the world. The underlying syndrome of CVD, atherosclerosis, is a complex disease process which involves lipid metabolism, inflammation, innate and adaptive immunity, and many other pathophysiological aspects. Furthermore, CVD is influenced by genetic as well as environmental factors. Early detection of CVD and identification of patients at risk are crucial to reduce the burden of disease and to allow personalized treatment. As established risk factors fail to accurately predict which part of the population is likely to suffer from the disease, novel biomarkers are urgently needed. Proteomics can play a significant role in identifying these biomarkers.
In this review, we describe the progress made in proteome profiling of the atherosclerotic plaque and several novel sources of potential biomarkers, including circulating cells and plasma extracellular vesicles (PEV). The importance of longitudinal biobanking in biomarker discovery is highlighted and exemplified by several plaque proteins identified in the biobank study Athero-Express. Finally, we discuss the post-translational modifications of proteins that are involved in atherosclerosis, which may become one of the foci in the ongoing quest for biomarkers through proteomics of plaque and other matrices relevant to the progression of atherosclerosis.
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Gamma irradiation of red blood cell (RBC) concentrates is routinely used to prevent transfusion-associated graft-versus-host disease (TA-GvHD). So far, the effects of ionizing radiation on RBC structure and function and especially the proteome are not fully understood.
RBC concentrates were irradiated with 30 Gy and stored for 1 or 15 days at 4 ± 2°C. Following cell lysis and hemoglobin depletion, 2D DIGE was used to examine the changes of the cytosolic RBC proteome. Significantly altered spots were analyzed using bottom-up proteomic approaches and selected marker proteins validated by western blotting.
Gamma irradiation was found to enhance conventional RBC storage lesions. Following 15 days of post-irradiation storage, the abundances of a total of 27 spots were significantly altered and three out of 13 identified proteins were selected and validated as potential marker proteins for the assessment of irradiation-induced cytosolic RBC lesions.
Gamma irradiation and subsequent ex vivo storage according to the European CE guidelines were found to affect RBC protein structures. The validated marker proteins can serve as a basis for the development of a screening assay to monitor the quality of irradiated RBC concentrates during ex vivo storage.
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Prostate cancer is one of the leading causes of male mortality in the Western world. Currently, radiation combined with hormone treatment is one of the principle curative regimes for localised disease. Significantly, of the patients treated this way approximately 25% subsequently experience disease recurrence and may require further treatment. At present, prostate specific antigen (PSA) is used to monitor treatment and a rising serum PSA level can be indicative of treatment failure. However, PSA is relatively insensitive as both transient increases in PSA without disease recurrence and disease recurrence without a rise in PSA occur. Hence, more effective biomarkers to monitor treatment and predict disease recurrence are needed. In this proof of principle study we used samples accrued under strict clinical trial governance and have applied a mass spectrometry based proteomic pipeline consisting of label free LC-MS biomarker discovery and multiple reaction monitoring confirmation strategy to identify a potential serum protein signature of disease recurrence. Ultimately, when extended and combined with validation studies using samples from other clinical trials we anticipate that this proteomics strategy will facilitate the development of a protein signature of significant clinical utility.
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There is a need to identify better glycan biomarkers for diagnosis, early detection and treatment monitoring in lung cancer using biofluids such as blood. Biofluids are complex mixtures of proteins dominated by a few high abundance proteins that may not have specificity for lung cancer. Therefore two methods for protein enrichment were evaluated; affinity capturing of IgG and enrichment of medium abundance proteins, thus allowing us to determine which method yields the best candidate glycan biomarkers for lung cancer.
N-glycans isolated from plasma samples from 20 cases of lung adenocarcinoma and 20 matched controls were analyzed using nLC-PGC-chip-TOF-MS. N-glycan profiles were obtained for five different fractions: total plasma, isolated IgG, IgG depleted plasma, and the bound and flow-through fractions of protein enrichment.
Four glycans differed significantly (FDR<0.05) between cases and controls in whole unfractionated plasma, while four other glycans differed significantly by cancer status in the IgG fraction. No significant glycan differences were observed in the other fractions.
These results confirm that the N-glycan profile in plasma of lung cancer patients is different from healthy controls and appears to be dominated by alterations in relatively abundant proteins.
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Purpose: Wisteria floribunda agglutinin-positive human Mac-2 binding protein (WFA+-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin-antibody sandwich immunoassay. In this study, we supplied recombinant WFA+-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA+-hM2BP quantification. Experimental design: The optimum conditions for producing recombinant WFA+-hM2BP were selected by cell glycome analysis based on a lectin microarray. Interlot variability of recombinant WFA+-hM2BP was determined using an antibody-overlay lectin microarray. Screening of anti-M2BP monoclonal antibody was completed by incorporating a WFA-antibody sandwich ELISA and an antibody-overlay lectin microarray. Results: The lectin microarray analysis revealed that human embryonic kidney 293 (HEK293) cells efficiently and stably produced WFA+-hM2BP in DMEM containing 10% FCS without any lot variation in the M2BP glycosylation level. A spiking experiment with recombinant WFA+-hM2BP was mostly effective for antibody screening. The reconstituted sandwich immunoassay was useful for the continuous quantification and cutoff index (COI) expression of serum WFA+-hM2BP. Conclusions and clinical relevance: The multiple use of lectin-assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.
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O-linked-β-N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of the 3′-hydroxyl groups of serine or threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. The cycling of this modification is regulated in response to nutrients, stress, and other extracellular stimuli by the catalytic activities of O-GlcNAc transferase and O-GlcNAcase. O-GlcNAc is functionally similar to phosphorylation and has been demonstrated to play critical roles in numerous biological processes, including cell signaling, transcription, and disease etiology. Since its discovery nearly thirty years ago, studies have demonstrated that the O-GlcNAc is highly abundant and widespread, like phosphorylation however, the development of methodologies to study O-GlcNAc at the site level has been challenging. Recently, a number of studies have overcome these challenges and describe new tagging, enrichment, and mass spectrometric-based approaches to study O-GlcNAc in terms of its site identification, stoichiometry, and dynamics on proteins. The development of these methods are key for elucidation of O-GlcNAc's functional crosstalk with phosphorylation and other PTMs, and will serve to provide the necessary information for the development of site-specific antibodies, which will aid in the determination of a particular protein's site specific function. In this review, we describe these methods and summarize results obtained from them demonstrating the roles of O-GlcNAc in diabetes, cancer, Alzheimer's, and in learning and memory, while also describing how these new strategies have implicated O-GlcNAc as a potential diagnostic for the screening of patients for prediabetes.
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Carbohydrate antigens are the most frequently and traditionally used biomarkers for cancer, such as CA19–9, CA125, DUPAN-II, AFP-L3, and many others. The diagnostic potential of them was simply based on the cancer-specific alterations of glycan structures on particular glycoproteins in serum/plasma. In spite of the facts that glycosylation disorders are feasible for cancer biomarkers and glycomic analysis technologies to explore them have been rapidly developed, it remains difficult to sensitively screen glycan structure changes on cancer-associated glycoproteins from clinical specimens. Moreover a lot of additional issues should be appropriately addressed for the clinical application of newly identified glycosylation biomarkers, including analytical throughput, quantitative confirmation of structural changes, and biological explanation for the alterations. In the last decade, significant improvement of mass spectrometric techniques is being made in the aspects of both hardware spec and pre-analytical purification procedures for glycoprotein analysis. Here we review potential approaches to perform comprehensive analysis of glycoproteomic biomarker screening from serum/plasma and to realize high throughput validation of site-specific oligosaccharide variations. The power and problems of mass spectrometric applications on the clinical use of carbohydrate biomarkers are also discussed in this review.
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Proteomics has fast become a standard tool in the life sciences, with increasingly sophisticated approaches and instruments delivering ever growing numbers of identified and quantified proteins. Yet despite the enormous technological progress, and the triumphant papers published on whole-cell proteomes being collected and analyzed, proteomics has so far failed to enter the clinic for routine applications. This is a peculiar contradiction, and one that warrants some closer study. I here argue that for proteomics to make a difference in the clinic, it needs to stop shirking responsibility, and to mature into an analytical, transparent and reproducible discipline that also invests in the consolidation of its technology rather than only focusing on the next big leap forward. A key enabling factor in this maturation process is quality control and quality assurance, with bioinformatics, in its least noticeable but most influential form, as a key underlying technology.
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Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown.
We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls.
A total of 197 out of ∼2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways.
Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs.
Epithelial ovarian carcinogenesis may occur de novo on the surface of ovarian mesothelial epithelial cells or from cells originating in other organs. Foreign müllerian cell intrusion into the ovarian environment has been hypothesized to explain the latter scenario. In this study, MALDI mass spectrometry (MS) profiling technology was used to provide molecular insights regarding these potentially different mechanisms
Using MALDI MS profiling, the molecular disease signatures were established in their molecular context. MALDI MS profiling was used on serous and endometrioid cancer biopsies to investigate cases of epithelial ovarian cancer. We then applied bioinformatic methods and identification strategies on the LC-MS/MS analyses of extracts from digested FFPE tissues. Extracts from selected regions (i.e., serous ovarian adenocarcinoma, fallopian tube serous adenocarcinoma, endometrioid ovarian cancer, benign endometrium and benign ovarian tissues) were performed, and peptide digests were subjected to LC-MS/MS analysis.
Comparison of the proteins identified from benign endometrium or three ovarian cancer types (i.e., serous ovarian adenocarcinoma, endometrioid ovarian adenocarcinoma and serous fallopian tube adenocarcinoma) provided new evidence of a possible correlation between the fallopian tubes and serous ovarian adenocarcinoma. Here, we propose a workflow consisting of the comparison of multiple tissues in their anatomical context in an individual patient.
The present study provides new insights into the molecular similarities between these two tissues and an assessment of highly specific markers for an individualized patient diagnosis and care.
The left ventricle (LV) responds to a myocardial infarction (MI) with an orchestrated sequence of events that results in fundamental changes to both the structure and function of the myocardium. This collection of responses is termed LV remodeling. Myocardial ischemia resulting in necrosis is the initiating event that culminates in the formation of an extracellular matrix (ECM)-rich infarct scar that replaces necrotic myocytes. While the cardiomyocyte is the major cell type that responds to ischemia, infiltrating leukocytes and cardiac fibroblasts coordinate the subsequent wound healing response. The matrix metalloproteinase (MMP) family of enzymes regulates the inflammatory and ECM responses that modulate scar formation. Matridomics is the proteomic evaluation focused on ECM, while degradomics is the proteomic evaluation of proteases as well as their inhibitors and substrates. This review will summarize the use of proteomics to better understand MMP roles in post-MI LV remodeling.
]]>Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood-based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer.
A SILAC-based quantitative proteomic approach was employed to identify secreted proteins that were differentially expressed between neoplastic and non-neoplastic gastric epithelial cells. Proteins from the secretome were subjected to SDS-PAGE and SCX-based fractionation, followed by mass spectrometric analysis on an LTQ-Orbitrap Velos mass spectrometer. Immunohistochemical labeling was employed to validate a subset of candidates using tissue microarrays.
We identified 2,205 proteins in the gastric cancer secretome of which 263 proteins were overexpressed >4-fold in gastric cancer-derived cell lines as compared to non-neoplastic gastric epithelial cells. Three candidate proteins, proprotein convertase subtilisin/kexin type 9 (PCSK9), lectin mannose binding 2 (LMAN2) and PDGFA associated protein 1 (PDAP1), were validated by immunohistochemical labeling.
We report here the largest cancer secretome described to date. The novel biomarkers identified in the current study are excellent candidates for further testing as early detection biomarkers for gastric adenocarcinoma.
Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine and cosmetics. Recently, Streptococcus zooepidemicus has been exploited in the fermentation industry to produce HA. Many studies showed that higher amounts of HA were produced under aerobic condition compared with anaerobic conditions. In order to explore the effect of oxygen on the HA synthesis in S. zooepidemicus, 2-DE was used to compare the proteomes of aerobically and anaerobically fermented bacteria to identify proteins, which might be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2-DE gels collected from three batches were compared and nine over-expressed proteins were observed in aerobically fermented bacteria. These proteins were identified by LC-MS/MS as dihydrolipoamide dehydrogenase, UDP-acetyl-glucosamine pyrophosphoylase, dihydrolipoamide-S-acetyltransferase, and acetoin dehydrogenase α and β chains, respectively. These up-regulated proteins were involved in acetoin dissimilation, the central carbon metabolism, and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are involved in central energy metabolism, acetoin reutilization, and HA biosynthesis to enhance the amount of acetyl-CoA as such more acetyl-CoA can be divergent from the central carbon metabolism to replenish acetyl-CoA for the HA synthesis.
To define mitochondrial protein markers related to liver cancer.
Mitochondrial subproteomes of 20 patient-derived liver carcinoma and tumor-free control tissues were performed by 2DE coupled with MALDI-TOF/TOF. The altered patterns of three identified proteins were validated by Western blot and immunohistochemistry.
The results showed that compared with tumor-free control samples, nine proteins were downregulated and six proteins were upregulated in carcinoma samples. The increased expression of Arg1 mRNA and protein was validated by Western blot, Q-RT-PCR, paraffin tissue microarray and immunohistochemistry. Furthermore, a literature review shows that Heat shock protein 10 (Hsp10), single-stranded DNA-binding protein (SSBP1), and peptidyl-prolyl cis-trans isomerase A (PPIA), which were identified as being increased in the tumor samples in this study, may be closely related to protein folding and translation.
These results show that in addition to changes in the signaling pathways, such as the Ras-Raf-MEK-ERK pathway, altered mitochondrial DNA replication and protein folding in liver cancer are also worth studying further. Collectively, these results suggest that specific mitochondrial proteins are uniquely susceptible to alterations in expression and carry implications for the investigation of their potential as therapeutic and prognostic markers. Further studies focusing on these proteins will be used to predict treatment response and reverse the apoptosis resistance.
Clinical application of biomarker candidates discovered by proteomic analysis is challenging. The purpose of this study was to standardize preanalytical conditions for measurement of serum levels of fibrinogen alpha C-chain 5.9 kDa fragment (FIC 5.9) and to test the diagnostic value of this peptide for detection of early hepatic fibrosis in patients with hepatitis C virus (HCV)-related chronic hepatitis.
Serum FIC 5.9 levels were measured by a sandwich ELISA. Effects on the serum FIC 5.9 level of temperature, the time between venipuncture and serum separation, and the types of collection tubes used were examined. The diagnostic value of serum FIC 5.9 as an early indicator of hepatic fibrosis due to HCV was then assessed.
FIC 5.9 was produced in a time- and temperature-dependent manner after venipuncture. Abnormal FIC 5.9 values were found in 89.5% of FI stage patients. Receiver operating characteristic analyses confirmed the superiority of FIC 5.9 over other conventional markers for early detection of fibrosis.
The serum FIC 5.9 level may be an early indicator of hepatic fibrosis in HCV-related chronic liver diseases. This study provides an example of a pipeline from biomarker discovery by proteome analysis to assay optimization and preliminary clinical validation.
We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection.
We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation (IVTT) system. Escherichia coli lysates were added to the plasma blocking buffer to reduce nonspecific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment.
We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins.
This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance.
In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT (HCIVT)) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu.
Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by HCIVT compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine.
HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than ten times higher than RRL, in both Western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array.
HCIVT is a robust in vitro transcription and translation system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.
A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis.
Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates.
The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts.
These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.
Clinical Proteomics has traveled a long way pinpointing potential biomarkers for a variety of diseases. However, the absence of clinical implementation of proteomics findings has led to a frank evaluation and reconsideration of applied practices in biomarker discovery, recruitment of technological tools for biomarker verification and generation of new guidelines for data reporting. Nevertheless, considering the need for vast clinical resources for biomarker validation, the frequent lack of clear definitions of contexts of use, in combination to the biomarker “high offer,” progress toward biomarker implementation will even more require the adoption of an extensive open-minded approach: disease-focused networks are needed to ensure rapid exchange of information, initiation of appropriate studies, parallel validation of multiple biomarkers and sharing of valuable clinical resources. This viewpoint article targets to reflect on these issues and advocates the added value of multidisciplinary networks in biomarker development using bladder cancer as a paradigm.
]]>Proteomics technologies are well suited for harnessing the immune response to tumor antigens for diagnostic applications as in the case of breast cancer. We previously reported a substantial impact of hormone therapy (HT) on the proteome. Here, we investigated the effect of HT on the immune response toward breast tumor antigens.
Plasmas collected 0–10 months prior to diagnosis of ER+ breast cancer from 190 postmenopausal women and 190 controls that participated in the Women's Health Initiative Observational Study were analyzed for the effect of HT on IgG reactivity against arrayed proteins from MCF-7 or SKBR3 breast cancer cell line lysates following extensive fractionation.
HT user cases exhibited significantly reduced autoantibody reactivity against arrayed proteins compared to cases who were Not Current users. An associated reduced level of IL-6 and other immune-related cytokines was observed among HT users relative to nonusers.
Our findings suggest occurrence of a global altered immune response to breast cancer-derived proteins associated with HT. Thus a full understanding of factors that modulate the immune response is necessary to translate autoantibody panels into clinical applications.
Although Crohn's colitis (CC) and ulcerative colitis (UC) share several clinical features, they have different causes, mechanisms of tissue damage, and treatment options. Therefore, the accurate diagnosis is of paramount importance in terms of medical care. The distinction between CC/UC is made on the basis of clinical, radiologic, endoscopic, and pathologic interpretations but cannot be differentiated in up to 15% of inflammatory bowel disease patients. Correct management of this “indeterminate colitis” depends on the accuracy of future, and yet not known, destination diagnosis (CC/UC).
We have developed a proteomic methodology that has the potential to discriminate between UC/CC. The histologic layers of 62 confirmed UC/CC tissues were analyzed using MALDI-MS for proteomic profiling.
A Support Vector Machine algorithm consisting of 25 peaks was able to differentiate spectra from CC and UC with 76.9% spectral accuracy when using a leave-20%-out cross-validation. Application of the model to the entire dataset resulted in accurate classification of 19/26 CC patients and 36/36 UC patients when using a 2/3 correct cutoff. A total of 114 peaks were found to have Wilcoxin rank sum p-values of less than 0.05.
This information may provide new avenues for the development of novel personalized therapeutic targets.
A number of studies have shown that the levels of some proteins in the aqueous humor (AH) are altered and correlate with the mechanisms or prognosis of many eye diseases. To identify the possible mechanisms that lead to the development of wet age-related macular degeneration (AMD), a proteomic analysis of the AH composition from wet AMD patients was performed and compared with that from non-AMD cataract patients.
Six wet AMD and six non-AMD cataract patients were enrolled. A proteomic approach which included two-dimensional electrophoresis coupled with MS and bioinformatics methods were used to identify AH proteins with altered expression in wet AMD compared with non-AMD patients. An ELISA was used to validate the proteomic results.
We separated 78 protein spots and identified 68 that were differently expressed in the wet AMD group and controls. Numerous proteins identified in this study are implicated in inflammation, apoptosis, angiogenesis, and oxidative stress.
The AH protein composition was significantly different between wet AMD and non-AMD patients. The proteins identified in this study may be potential biomarkers of wet AMD development and might play a role in the mechanisms of wet AMD.
Studies from our laboratory have reported 14 tumor antigens that elicit an autoantibody response in patients with cancer of the gingivobuccal complex (GBC) In this study, utility of the autoantibody response has been evaluated for prognosis of cancer of the GBC.
Autoantibody response was evaluated using immunoproteomics and the prognostic significance was assessed by Kaplan-Meier survival and multivariate analysis.
Autoantibody response against α-enolase isoforms a, b, and c and Hsp70 was detected in 27, 53, 64, and 26% of the 78 patients, respectively. Patients positive for autoantibody response to α-ENO and Hsp70 individually and in combination, showed significantly reduced disease-free survival (DFS) compared to those who do not show autoantibody response to either of them. Further the patients, who exhibit autoantibody response to α-ENO and Hsp70 in combination with nodal involvement and/or differentiation status, have significantly lowered DFS. The relative risk of recurrence is 3.41 for patients who exhibit autoantibody response to both the antigens.
Autoantibody response against α-ENO and Hsp70 provides an additional parameter and may be utilized along with nodal involvement and differentiation status for better prognosis of cancer of GBC.
The process of new blood vessel formation from pre-existing ones is called angiogenesis. Beyond playing a critical role in the physiological development of the vascular system, angiogenesis is a well-recognised hallmark of cancer. Unbiased system-wide approaches are required to complement the current knowledge, and intimately understand the molecular mechanisms regulating this process in physiological and pathological conditions. In this review we describe the cellular and molecular dynamics regulating the physiological growth of vessels and their deregulation in cancer, survey in vitro and in vivo models currently exploited to investigate various aspects of angiogenesis and describe state-of-the-art and most widespread methods and technologies in MS shotgun proteomics. Finally, we focus on current applications of MS to better understand endothelial cell behaviour and propose how modern proteomics can impact on angiogenesis research.
]]>Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics.
Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS.
Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta.
Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification.
Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample.
The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer.
Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer.
Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets.
We have developed a new method for rapid analysis of a specific region on formalin fixed and paraffin embedded (FFPE) tissue sections. This method combines advantages of direct tissue MS analysis keeping histological information and conventional proteomics approaches for confident identification of proteins in complex sample.
After histological annotation, heat-induced antigen retrieval is performed on FFPE tissue. Using a chemical inkjet printer, trypsin is deposited on discrete regions of less than 1 mm2. After protein digestion, a liquid extraction is performed to retrieve all the peptides. Data coming from identification of proteins in cancer and benign region are compared.
In total, 3649 unique peptides were identified (with a peptide strict false discovery rate less than 1%) corresponding to 983 and 792 nonredundant protein groups identified in benign and cancer region, respectively. A total of 123 protein groups are found only in cancer region and 315 are specific to the benign part. From these data, it has been possible to obtain different important signaling pathways involved in cancer processes and some proteins already known as biomarkers.
This new approach using a combination of localized on-tissue protein digestion and liquid microextraction followed by LC-MS/MS analysis is useful for advancing our understanding of cancer biology. It is a rapid and innovative technique that will contribute positively to clinical proteomics.
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.
]]>In the recent past, the potential suitability of fixed samples to 2D-DIGE studies has been demonstrated on model tissues, but not on “real-world” archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2D-DIGE on samples retrieved from hospital tissue repositories.
Diseased and normal tissue samples (namely, human gastric adenocarcinoma and normal gastric tissue, human lung neuroendocrine tumors, canine mammary tubulo-papillary carcinoma and normal mammary tissue, sheep liver with cloudy swelling degeneration and normal liver tissue) were retrieved from human and veterinary biorepositories and subjected to full-length protein extraction, cyanine labeling, 2D-DIGE separation, image analysis, MS analysis, and protein identification.
Archival samples could be successfully subjected to 2D-DIGE, providing maps of satisfactory resolution, although with varying pattern complexity (possibly influenced by preanalytical variables). Moreover, differentially expressed protein identities were consistent with the disease biology.
2D-DIGE can support biomarker discovery and validation studies on large sample cohorts. In fact, although some information complexity is lost when compared to fresh-frozen tissues, their vast availability and the associated patient information can considerably boost studies suffering limited sample availability or involving long-distance exchange of samples.
Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.
]]>Protein profiling of formalin-fixed paraffin-embedded (FFPE) tissues has enormous potential for the discovery and validation of disease biomarkers. The aim of this study was to systematically characterize the effect of length of time of storage of such tissue blocks in pathology archives on the quality of data produced using label-free MS.
Normal kidney and clear cell renal cell carcinoma tissues routinely collected up to 10 years prior to analysis were profiled using LC-MS/MS and the data analyzed using MaxQuant. Protein identities and quantification data were analyzed to examine differences between tissue blocks of different ages and assess the impact of technical and biological variability.
An average of over 2000 proteins was seen in each sample with good reproducibility in terms of proteins identified and quantification for normal kidney tissue, with no significant effect of block age. Greater biological variability was apparent in the renal cell carcinoma tissue, possibly reflecting disease heterogeneity, but again there was good correlation between technical replicates and no significant effect of block age.
These results indicate that archival storage time does not have a detrimental effect on protein profiling of FFPE tissues, supporting the use of such tissues in biomarker discovery studies.
Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancers, and has a worse prognosis compared with hormone receptor-positive disease. Its unfavorable outcome and the lack of hormonal receptors determine the use of adjuvant chemotherapy as part of the standard treatment for these tumors, although several studies have documented that the current standard combination chemotherapy is suboptimal. Therefore, a new functional taxonomy of breast cancer and new targets for therapeutic development are urgently needed.
In this study, we have analyzed the proteome of TNBC applying a high-throughput proteomics approach to routinely archived formalin-fixed, paraffin-embedded tumor tissues.
We have been able to identify and quantify more than 1000 protein groups. Some of these proteins are of outstanding interest in the biology and clinical management of this disease, such as CD44 and PARP1. Moreover, we have characterized some signaling pathways that could be related to TNBC genesis and development.
Our results open up new avenues for the use of proteomics technologies in clinically relevant studies using archival samples. Shotgun LC-MS/MS studies could serve to discover new biomarkers and may provide clues to the genesis of TNBC and underlying molecular alterations.