Proteomic analysis of colorectal cancer: discovering novel biomarkers

Colorectal cancer is one of the most common cancers in the Western world. When detected at an early stage, the majority of cancers can be cured with current treatment modalities. However, most cancers present at an intermediate stage. The discovery of sensitive and specific biomarkers has the potential to improve preclinical diagnosis of primary and recurrent colorectal cancer, and holds the promise of prognostic and therapeutic application. Current biomarkers such as carcinoembryonic antigen lack sensitivity and specificity for general population screening. This review aims to highlight the role of current proteomic technologies in the discovery and validation of potential biomarkers with a view to translation to the clinic.     

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.     

Proteomic approach to aging research

The scope of the current paper is to review existing and potential applications of proteomic analysis to aging research. The focus will lie on the unique opportunities of high-throughput studies for uncovering specific alterations in protein expression, protein complexes or protein modifications caused by biological aging. The result of such studies will outline aging phenotypes and potentially indicate pathways involved in the pathogenesis of age-associated disfunctions. Specific attention is paid to the illustrations of successful applications of proteomic technologies and potential applications of new proteomic concepts to biogerontological studies.     

Tissue and serum proteomic profiling for diagnostic and prognostic bladder cancer biomarkers

A panel of biomarkers for the early detection of bladder cancer has not yet been identified. Many different molecules, including DNA, RNA or proteins have been reported but none have provided adequate sensitivity for a single-tier screening test or a test to replace cystoscopy. Therefore, multimarker panels are discussed at present to give a more-precise answer to the biomarker quest. Mass spectrometry or 2D gel-electrophoresis have evolved greatly within recent years and are capable of analyzing multiple proteins or peptides in parallel with high sensitivity and specificity. However, transmission of screening results from one laboratory to another is still the main pitfall of those methods; a fact that emphasizes the need for consistent and standardized procedures as suggested by the Human Proteome Organization (HUPO). In this article, recent results in screening approaches and other proteomic techniques used for biomarker evaluation in bladder cancer are discussed with a focus on serum and tissue biomarkers.     

Advanced proteomic technologies for cancer biomarker discovery

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.     

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS^E proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.     

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.     

Type 1 diabetes: entering the proteomic era

During the last decade, a major breakthrough in the field of proteomics has been achieved. This review describes available techniques for proteomic analyses, both gel and non-gel based, particularly concentrating on relative quantification techniques. The principle of the different techniques is discussed, highlighting the advantages and drawbacks of recently available visualization methods in gel-based assays. In addition, recent developments for quantitative analysis in non-gel-based approaches are summarized. This review focuses on applications in Type 1 diabetes. These mainly include proteomic studies on pancreatic islets in animal models and in the human situation. Also discussed are mass spectrometry-based studies on T-cells, and studies on the development of diagnostic markers for diabetic nephropathology by capillary electrophoresis coupled to mass spectrometry.     

大鼠海马的表达蛋白质组学实验研究

目的：用蛋白质组学方法初步分析大鼠海马蛋白质的表达。方法：提取大鼠海马蛋白质样品后,用双向凝胶电泳对其分离,经考马斯亮蓝染色后,产生大鼠海马蛋白质双向凝胶电泳图谱。从凝胶上切割分离的蛋白质,经胰蛋白酶胶内酶解,通过基质辅助激光解吸／电离飞行时间质谱（MALDI-TOF-MS）对酶解后的肽段进行分析。根据肽段质谱数据,经数据库（NCBI）检索,对蛋白质进行鉴定。结果：鉴定了37种具有明确功能的蛋白质,它们分别属于代谢酶、细胞骨架蛋白、热休克蛋白、抗氧化蛋白、信号传导蛋白、蛋白酶体相关蛋白、神经元特异蛋白及神经胶质蛋白。另外,鉴定了3种未知功能蛋白。结论：为建立大鼠海马蛋白质组数据库提供必要的资料,为在大鼠模型上研究神经疾病发病机理奠定基础。     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging

3-Nitrotyrosine (3-NT) is a useful biomarker of increasing oxidative stress and protein nitration during biological aging. The proteomic analysis of cerebellar homogenate from Fisher 344/Brown Norway (BN/F1) rats shows an age-dependent increase in protein nitration, monitored by western-blot analysis after two-dimensional gel electrophoresis (2DE), mainly in the acidic region. Analysis of in-gel digests by nanoelectrospray (NSI)-MS/MS resulted in the identification of 16 putatively nitrated proteins. The selective isolation of nitrated proteins using immunoprecipitation, followed by SDS-PAGE and in-gel digest/NSI-MS/MS analysis led to the identification of 22 putatively nitrated proteins, of which 7 were identical to those detected after 2DE. When proteins were separated by solution isoelectrofocusing and analyzed by NSI MS/MS, we obtained MS/MS spectra of 3-NT containing peptides of four proteins - similar to ryanodine receptor 3, low density lipoprotein related receptor 2, similar to nebulin-related anchoring protein isoform C and 2,3 cyclic nucleotide 3-phosphodiesterase. Although the functional consequences of protein nitration for these targets are not yet known, our proteomic experiments serve as a first screen for the more targeted analysis of nitrated proteins from aging cerebellum for functional characterization.     

Proteomic approaches for studying chemoresistance in cancer

The role of various proteins involved in drug resistance in tumor cells is discussed in this review. Two types of studies are covered: those performed in the preproteomics era and those carried out with modern proteomic tools, namely 2D (electrophoretic) maps and 2D chromatography. In the preproteomic studies, one protein had generally been held responsible for a given chemoresistance. However, analysis via proteomic tools may reveal entire sets of proteins that are up- or downregulated (or switched on/off) in chemoresistant tumor cell lines compared with parental tumor lines. Therefore, it appears more realistic to expect that exposure of cells to drugs results in the activation of different mechanisms of resistance. Such investigations have led to the broadly shared opinion that exposure of cells to drugs results in the activation of different mechanisms of resistance, and that a specific drug-resistant phenotype consists of several molecular mechanisms that are simultaneously active. The proteomic papers reviewed clearly support the hypothesis that many metabolic pathways are affected during the resistance process. Although the modulation of expression levels of such proteins is not clear proof of their role in drug resistance per se, at least some of the themes are very likely to be involved in the resistance phenotype, and thus may be potential targets for new drugs. It is hoped that this review will bring new insight in this field and will stimulate novel and deeper searches with proteomic tools (including prefractionation of subcellular organelles, such as nuclei, to bring to the fore low-abundance proteins that might be responsible for the onset of drug resistance).     

Proteomic analysis of cellular signaling

Protein phosphorylation events are key regulators of cellular signaling processes. In the era of functional genomics, rational drug design programs demand large-scale high-throughput analysis of signal transduction cascades. Significant improvements in the area of mass spectrometry-based proteomics have provided exciting opportunities for rapid progress toward global protein phosphorylation analysis. This review summarizes several recent advances made in the field of phosphoproteomics with an emphasis placed on mass spectrometry instrumentation, enrichment methods and quantification strategies. In the near future, these technologies will provide a tool that can be used for quantitative investigation of signal transduction pathways to generate new insights into biologic systems.     

Proteomic analysis at the bedside: early detection of cancer

Proteomic technologies promise to accelerate rapidly a new era in molecular medicine, especially in the detection and discovery of disease-related biomarkers. These technologies have no bigger impact than in the field of human cancer research. Beyond lifestyle-associated prevention strategies, early detection of cancer has the most profound impact on the ultimate course of the disease: the earlier the cancer is detected, the better the prognosis. Today, new proteomic technologies are being used to discover new diagnostic and prognostic biomarkers for the early detection and treatment of cancer that will have important implications at the bedside.     

癌症差异蛋白质组学研究中样品分离和鉴定分析技术

随着人类基因组测序的完成,癌症研究的重点从基因组学转移到蛋白质组学研究中。癌症研究中的差异蛋白质组学技术也飞速发展,包括癌症样品制备、分离,蛋白质鉴定分析、蛋白质组定量研究和翻译后修饰研究等。这些技术极大地推动了与癌症相关的差异蛋白质组学研究,使蛋白质组学在癌症早期诊断、治疗,监测以及发现新药物治疗靶标方面发挥更大的作用。本文主要综述了近年来癌症差异蛋白质组学研究中样品分离和鉴定分析技术。     

Proteomic analysis of traumatic brain injury: the search for biomarkers

Although there are a number of causes of traumatic brain injury (TBI), the armed conflict in Iraq and Afghanistan has brought this disorder to the attention of the global community. A biomarker that would enable army medics to rapidly diagnose the severity of TBI on the battle-field would be a huge asset. Unfortunately, the study of TBI has not historically attracted the proteomic research community’s interest as other disorders have, such as cancer. On the positive side, however, many of the analytical and technological challenges that were overcome in the development of biofluid proteomic methods are now being applied to the study of TBI. In this review, we discuss and highlight select examples of discovery-driven proteomic studies focused on finding effective biomarkers for TBI.     

A potential biomarker of kidney damage identified by proteomics: preliminary findings

4-Aminophenol (4-AP) and d-serine are established rodent nephrotoxins that selectively damage renal proximal tubules. In an attempt to understand the mechanism of action of these toxicants in greater detail, a high throughput proteomics approach was used to profile protein changes in the plasma of animals treated with these compounds. Male Fischer 344 and Alderley Park rats were treated with increasing doses of 4-AP or d-serine and plasma samples were collected over time. Control groups received either saline or the non-toxic enantiomer, l-serine. Using high throughput two-dimensional gel analysis, a number of plasma proteins showing dose- and time-dependent regulation were identified. One toxicity-associated plasma protein was identified as the cellular enzyme fumarylacetoacetate hydrolase (FAH), which is known to be required for tyrosine metabolism. The FAH gene is mutated in the human genetic disorder type I tyrosinaemia, which is associated with liver and kidney abnormalities and neurological disorders. FAH was elevated in the plasma of animals treated with 4-AP and d-serine at early time points and returned to baseline levels after 3 weeks. The protein was not elevated in the plasma of control animals or those treated with l-serine. The presence of FAH in plasma is intriguing as it is normally a cellular enzyme with no known function in plasma. It is possible that 4-AP and d-serine may work through a previously unknown mechanism in the kidney via regulation of tyrosine metabolism or FAH activity. Therefore, FAH may function in a fashion analogous to the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes that are used to measure liver injury. The link between kidney toxicants and inherited tyrosinaemia also raises the possibility that FAH may be a marker of kidney toxicity in humans. These observations highlight the value of proteomics in identifying new biomarkers and providing new unprecedented insights into complex biological mechanisms.     

A potential biomarker of kidney damage identified by proteomics: preliminary findings

4-Aminophenol (4-AP) and d-serine are established rodent nephrotoxins that selectively damage renal proximal tubules. In an attempt to understand the mechanism of action of these toxicants in greater detail, a high throughput proteomics approach was used to profile protein changes in the plasma of animals treated with these compounds. Male Fischer 344 and Alderley Park rats were treated with increasing doses of 4-AP or d-serine and plasma samples were collected over time. Control groups received either saline or the non-toxic enantiomer, l-serine. Using high throughput two-dimensional gel analysis, a number of plasma proteins showing dose- and time-dependent regulation were identified. One toxicity-associated plasma protein was identified as the cellular enzyme fumarylacetoacetate hydrolase (FAH), which is known to be required for tyrosine metabolism. The FAH gene is mutated in the human genetic disorder type I tyrosinaemia, which is associated with liver and kidney abnormalities and neurological disorders. FAH was elevated in the plasma of animals treated with 4-AP and d-serine at early time points and returned to baseline levels after 3 weeks. The protein was not elevated in the plasma of control animals or those treated with l-serine. The presence of FAH in plasma is intriguing as it is normally a cellular enzyme with no known function in plasma. It is possible that 4-AP and d-serine may work through a previously unknown mechanism in the kidney via regulation of tyrosine metabolism or FAH activity. Therefore, FAH may function in a fashion analogous to the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes that are used to measure liver injury. The link between kidney toxicants and inherited tyrosinaemia also raises the possibility that FAH may be a marker of kidney toxicity in humans. These observations highlight the value of proteomics in identifying new biomarkers and providing new unprecedented insights into complex biological mechanisms.     

Proteomic analysis of tissue samples in translational breast cancer research

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies – based on the analysis of clinically relevant samples – that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.