Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience: exemplified in amyotrophic lateral sclerosis biomarker discovery research

Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neurodegenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).     

Proteomics of human cancer tissues and cells

Proteomic analysis of cancer tissues and cells provides valuable information to identify promising targets for cancer diagnosis, prognosis and therapy. Novel strategies have emerged to optimize the workflow of tissue procurement, and tissue and cell selection, and to improve protocols for the extraction of protein from fresh, frozen and paraffin-embedded tissue. Moreover, in the context of advanced approaches to proteomics, mass spectrometry and array-based technologies strongly contribute to protein profiling of cancer tissues and cells.The focus of this review is the methods by which all the steps of a proteomic investigation on human-cancer tissue (from choice of the experimental model to validation of candidate biomarkers) should be performed, paying particular attention to recently developed strategies. The review also presents an overview of the most recent high-throughput proteomic studies in cancer research.     

From Chemistry to Translational Medicine: The Application of Proteomics to Cancer Biomarker Discovery and Diagnosis

The study of complex protein mixtures and their interactions in cells and tissues has been difficult due to the tedious process involved in their characterization and analysis. The recent emergence of fast‐evolving and state‐of‐the‐art proteomics methodologies has provided a rapid and scalable platform for understanding the comprehensive proteome profiles from complex whole tissues or cells of various biological sources. Therefore, proteomics has been increasingly valuable to examine real‐time changes in protein expression of various tissues or body fluids from patients with various diseases, especially cancer, resulting in the identification of clinically useful biomarkers for diagnosis, prognosis and disease staging. In this review, we focus on potential biomarkers for (1) Helicobacter pylori‐associated gastric cancer, (2) hepatocellular carcinoma (HCC), and (3) renal cell carcinoma (RCC). In addition to the conventional gel‐based proteomics (1‐D or 2‐D gels), we have utilized a more advanced proteomic approach by incorporating stable isotope dimethyl labelling and shotgun proteomics strategy in combination with nanoliquid chromatography and tandem mass spectrometry (nanoLC‐MS/MS) to better characterize the biomarkers in several cancer tissues. By establishing a high‐throughput proteomics platform based on multiple reaction monitoring (MRM), we have successfully detected and analyzed potential protein markers at low concentrations in various normal and tumor tissues. This platform not only highlights the utility of proteomics for biomarker discovery but also can be uniquely applied to disease‐oriented translational medicine for diagnosis of diverse types of cancers and other diseases.     

Monoclonal antibody proteomics: discovery and prevalidation of chronic obstructive pulmonary disease biomarkers in a single step

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.     

The discovery and validation of colorectal cancer biomarkers

Colorectal cancer is currently the third most common malignancy in the world. Patients have excellent prognosis following surgical resection if their tumour is still localized at diagnosis. By contrast, once the tumour has started to metastasize, prognosis is much poorer. Accurate early detection can therefore significantly reduce the mortality from this disease. However, current tests either lack the required sensitivity and selectivity or are costly and invasive. Improved biomarkers, or panels of biomarkers, are therefore urgently required. We have addressed current screening strategies and potential protein biomarkers that have been proposed. The role of both discovery and hypothesis-driven proteomics approaches for biomarker discovery and validation is discussed. Using such approaches we show how multiple reaction monitoring (MRM) can be successfully developed and used for quantitative multiplexed analysis of potential faecal biomarkers.     

Proteomic profiling of protein corona formed on the surface of nanomaterial

The formation of protein coronas on nanomaterial will significantly alter the surface properties of nanomaterial in biological systems and subsequently impact biological responses including signaling, cellular uptake, transport, and toxicity etc. It is of critical importance to understand the formation of protein coronas on the surface of nanomaterial. Analytical techniques, especially mass spectrometry-based proteomics methods, are playing a key role for the qualitative and quantitative analyses of protein coronas on nanomaterial. In this review, the proteomic approaches developed for the characterization of protein coronas on various nanomaterials are introduced with the emphasis on the mass spectrometry-based proteomic strategies.     

基于质谱技术蛋白质定量方法的研究进展

质谱技术已经成为目前蛋白质鉴定的重要工具。定量分析细胞内蛋白质组的动态变化,是当前研究蛋白质功能、揭示细胞生物机理、寻找疾病蛋白标记物和药物靶标的迫切需要。本文综述了基于质谱技术蛋白质定量的策略、方法和应用等方面近年来的进展,评述了几种蛋白质质谱定量方法的特点和应用潜力。     

Ovarian Cancer: Biomarker Proteomic Diagnosis in Progress

Ovarian cancer lacks clear syndromes at an early stage and could result in serious problem in woman's health status. The current diagnostic approach relies on physical examination, ultrasound examination, and blood test for CA125. These approaches could not diagnose early stage ovarian cancer with high sensitivity and specificity. The present paper reviewed the efforts in screening the proteomic biomarkers for ovarian cancer. The sources of biomarkers were discussed. Then, the current techniques in proteomics were introduced. Finally, the biomarkers for ovarian cancer were summarized.     

基于质谱检测的相关色谱技术在阿尔茨海默病诊断中的应用

随着世界老年人口的急速增长,阿尔茨海默病发病人数也逐年增多,已成为继心脑血管疾病和恶性肿瘤之后威胁人类健康的“第三大杀手”。疾病的诊断和治疗同等重要,阿尔茨海默病诊断通常依靠典型的临床特征、神经影像技术以及检测疾病相关的生物标志物等。近些年来蛋白质组学和质谱技术迅速发展,可以利用这些技术寻找到与疾病相关的特异性的蛋白质分子作为早期诊断的生物标志物。本文就此进行了综述,主要包括基于蛋白质组学的诊断标志物的筛选和基于质谱检测的色谱技术在阿尔茨海默病诊断中的应用,引用文献34篇。     

Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposes

The introduction of fast, sensitive, and robust techniques for proteomic analysis into clinical practice represents a major step toward a new diagnostic approach of body fluids. In addition, proteomics emerges as a key technology for the discovery of disease biomarkers in various body fluids. However, even in relatively protein-deprived body fluids such as urine, the complexity and wide dynamic range of protein expression pose a considerable challenge to both separation and identification technologies. In the present review we discuss from a clinical point-of-view recent advances of the use of proteomics in clinical diagnosis as well as therapy evaluation. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS) and discuss CE-MS from an application point of view evaluating its merits and vices with regard to biomarker discovery. This review further presents examples of clinical applications of CE-MS for detection and identification of biomarkers in urine.     

A comprehensive and non‐prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine

Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre‐fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin‐yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high‐coverage identification of peptide mixtures. In this study, we adopted a strategy without pre‐fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin‐yang MDLC methods for peptide fractionation followed by identification using a linear ion trap‐orbitrap (LTQ‐Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non‐redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane‐related proteins. In addition, the lysosomal as well as kidney‐associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery. Copyright © 2010 John Wiley & Sons, Ltd.     

Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care

Ovarian cancer is asymptomatic in the early stages and most patients present with advanced levels of disease. The lack of cost-effective methods that can achieve frequent, simple and non-invasive testing hinders early detection and causes high mortality in ovarian cancer patients. Here, we report a simple and inexpensive microchip ELISA-based detection module that employs a portable detection system, i.e., a cell phone/charge-coupled device (CCD) to quantify an ovarian cancer biomarker, HE4, in urine. Integration of a mobile application with a cell phone enabled immediate processing of microchip ELISA results, which eliminated the need for a bulky, expensive spectrophotometer. The HE4 level detected by a cell phone or a lensless CCD system was significantly elevated in urine samples from cancer patients (n = 19) than healthy controls (n = 20) (p < 0.001). Receiver operating characteristic (ROC) analyses showed that the microchip ELISA coupled with a cell phone running an automated analysis mobile application had a sensitivity of 89.5% at a specificity of 90%. Under the same specificity, the microchip ELISA coupled with a CCD had a sensitivity of 84.2%. In conclusion, integration of microchip ELISA with cell phone/CCD-based colorimetric measurement technology can be used to detect HE4 biomarker at the point-of-care (POC), paving the way to create bedside technologies for diagnostics and treatment monitoring.     

Application of capillary electrophoresis for the early diagnosis of cancer

Early diagnosis is the key to the effective treatment of cancer. The detection of cancer biomarkers plays a critical role not only in cancer early diagnosis, but also in classification and staging tumor progression, or assessment prognosis and treatment response. Currently, various molecular diagnostic techniques have been developed for cancer biomarker studies, with many of the more effective approaches requiring a separation step before detection. Capillary electrophoresis (CE) can perform rapid and efficient separation with small samples, which is well-suited for analysis of both small- and macro- molecule biomarkers in complex samples. CE has different separation modes and can couple to different detectors into a variety of platforms, such as conducting studies on DNA/ RNA point mutation, protein misexpression, and metabolite abnormality. Similarly, microchip capillary electrophoresis (MCE) appears as a very important biomarker screening platform with the merits of high throughput, integration, and miniaturization, which makes it a promising clinical tool. By hyphenated different detectors, or integrated with immunoassay, PCR/LDR and related technologies, MCE can be constructed into diverse platforms used in genomics, proteomics, and metabolomics study for biomarkers discovery. The multiplex biomarker screening approach via CE- or MCE-based platforms is becoming a trend. This paper focuses on studies of cancer biomarkers via CE/MCE platforms, based on the studies published over the past 3 years. Some recent CE applications in the field of cancer study, such as cancer theranostics, are introduced.     

Quantitative proteomics for cancer biomarker discovery

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), 1?O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.     

Mass spectrometry in biodefense

Potential agents for biological attacks include both microorganisms and toxins. In mass spectrometry (MS), rapid identification of potential bioagents is achieved by detecting the masses of unique biomarkers, correlated to each agent. Currently, proteins are the most reliable biomarkers for detection and characterization of both microorganisms and toxins, and MS-based proteomics is particularly well suited for biodefense applications. Confident identification of an organism can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass spectra. In bottom-up proteomics, rapid digestion of intact protein biomarkers is again followed by MS/MS to provide unambiguous bioagent identification and characterization. Bioinformatics obviates the need for culturing and rigorous control of experimental variables to create and use MS fingerprint libraries for various classes of bioweapons. For specific applications, MS methods, instruments and algorithms have also been developed for identification based on biomarkers other than proteins and peptides.     

MALDI tissue imaging: from biomarker discovery to clinical applications

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.     

Breath biosensing: using electrochemical enzymatic sensors for detection of biomarkers in human breath

Detection of biomarkers for disease by noninvasive methods is critical for the early diagnosis and screening of disease, enabling prompt treatment. Breath biosensors are a viable option as the exhaled breath contains several biomarkers linked to lung cancer, oxidative stress, diabetes, and other diseases. Breath analysis has been achieved by advanced analytical techniques such as gas chromatography and infrared spectroscopy. However, electrochemical enzymatic breath biosensors offer a cost-effective, sensitive platform for biomarker detection without complex analysis and interpretation by trained laboratory personnel. This review aims to summarize recent advances in the field of electrochemical enzymatic breath biosensors and offer future opportunities from other applications of nonelectrochemical enzymatic breath biosensors.     

Expanded metabolomics approach to profiling endogenous carbohydrates in the serum of ovarian cancer patients

We applied hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry to the quantitative analysis of serum from 58 women, including ovarian cancer patients, ovarian benign tumor patients, and healthy controls. All of these ovarian cancer and ovarian benign tumor patients have elevated cancer antigen 125, which makes them clinically difficult to differentiate the malignant from the benign. All of the 16 endogenous carbohydrates were quantitatively detected in the human sera, of which, eight endogenous carbohydrates were significantly different (P‐value < 0.05) between the ovarian cancer and healthy control. According to the receiver operating characteristic curve analysis, arabitol was the most potentially specific biomarker for discriminating ovarian cancer from healthy control, having an area under the curve of 0.911. A panel of metabolite markers composed of maltose, maltotriose, raffinose, and mannitol was selected, which was able to discriminate the ovarian cancer from the benign ovarian tumor counterparts, with an area under concentration‐time curve value of 0.832. Endogenous carbohydrates in the expanded metabolomics approach after the global metabolic profiling are characterized and are potential biomarkers for the early diagnosis of ovarian cancer.     

肽组学样品前处理方法与技术进展

肽组学作为蛋白质组学研究领域的一个重要分支,在生物标志物发现、疾病早期诊断、生物制药等领域有着广阔的应用前景。在肽组学研究过程中,样品前处理是首要环节。本文对肽组学样品前处理方法与技术进行了综述,内容涉及超滤技术、有机溶剂沉淀方法、固相萃取技术等。样品中大量高丰度蛋白质的存在是肽组学发展面临的最大挑战,因此迫切需要发展快速、有效、高通量、自动化的样品前处理方法和技术。