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.     

Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling

Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to the target species. External calibration was employed as a quantification technique. The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column. The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme and bovine serum albumin.     

基于稳定同位素标记的蛋白质组学定量方法研究进展

定量蛋白质组学已经成为后基因时代的重要研究方向之一。目前该领域的研究主要采用无标记定量方法和稳定同位素标记定量法。其中,基于稳定同位素标记的蛋白质组定量方法发展非常迅速,已为生命科学研究提供了重要的技术支撑。本文分析了基于稳定同位素标记的蛋白质组学定量方法,包括相对定量方法和绝对定量方法,并对其发展进行了展望。     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

基于稳定同位素标记与质谱分析的蛋白质定量算法研究进展

蛋白质定量研究已成为蛋白质组学的热点,它是疾病相关生物标志物发现的重要途径.基于稳定同位素标记的质谱分析技术是蛋白质定量最常用的方法之一.随着实验方法的发展和改进,定量数据处理方法也在不断更新与完善.一般来说,定量数据处理包括四步:搜库鉴定、图谱定量信息提取与计算、肽段丰度比计算和蛋白质丰度比计算及差异显著性分析,其中后三步是数据处理的核心.目前,后三步中每步都有多种可选算法,这些算法一般都是针对特定实验技术而提出的,缺乏深入的工作对它们进行系统比较和优化.为此,在总结目前主要实验技术方法的基础上,论述了定量算法的现状和存在问题,并针对一些问题提出了可行的解决办法.     

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low‐abundance prey proteins after intracellular photocrosslinking and prey–bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease–chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.     

The expanding field of SILAC

Stable isotope labeling by amino acids (SILAC) metabolically encodes cell populations for protein quantification by mass spectrometry. SILAC was introduced in 2002 and the field of mass spectrometry based proteomics has changed dramatically over the last decade. Increased sensitivity and speed of mass spectrometry instruments coupled with significantly improved mass resolution and precision have led to much higher rates of peptide identification and deeper coverage of proteomic samples. Several proteomics approaches are now available for quantifying proteins and their post-translational modifications, each with their strengths and weaknesses. The simplicity and robustness of SILAC have led to its widespread adoption and new applications have emerged that play to its particular strengths as a metabolic labeling approach.     

Applications of stable isotope dimethyl labeling in quantitative proteomics

Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.     

比较蛋白质组学研究中的稳定同位素标记技术

比较蛋白质组学是指在蛋白质组学水平上研究正常和病理情况下细胞或组织中蛋白质表达变化,以期发现具有重要功能的生物标识物,为疾病的早期诊断提供依据。近年来它正成为蛋白质组学研究的热点和发展趋势。比较蛋白质组学的研究方法和策略有多种,本文就最近几年来稳定同位素标记技术(体内代谢标记技术和体外化学标记技术)在比较蛋白质组学研究中的进展进行综述。     

Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS

A multifunctional isothiocyanate-based isotope labeling reagent, [d ₀]-/[d ₆]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b₁ ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b₁ ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.     

N-Glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans

N-Linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95 % derivatization was achieved in 30 min and the labeled glycans, when kept at ?20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC–MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

The potential of inorganic mass spectrometry in mineral and trace element nutrition research

Over the past two decades, new applications of inorganic mass spectrometry have been made possible by the use of stable isotopes as tracers in studies of mineral and trace element metabolism in man. Stable isotope techniques and radioisotope methods are the only reliable tools available for determination of the absorption, retention, or utilization of a nutrient by the human body. Recent developments in inorganic mass spectrometry might open new perspectives as progress in this field of research depends mainly on improving existing stable isotope techniques and on developing novel concepts. By improving precision in isotope analysis, isotope doses in experiments on man can be reduced to physiologically more meaningful levels. This will also enable reduction of the (often substantial) costs of isotopically labeling a nutrient in a test meal. Improvements in the mass spectrometric sensitivity will enable the development of new tracer techniques that have the potential to provide the information required by: 1. governmental institutions for designing food fortification programs; 2. the food industry for developing nutrient-fortified food products; and 3. public health authorities for establishing reliable dietary recommendations for intake of inorganic nutrients. In this context the current scope and limitations of thermal ionization mass spectrometry, inductively coupled mass spectrometry, accelerator mass spectrometry, and resonance ionization mass spectrometry are evaluated. Iron isotopic variations in the human body are discussed as a possible source of bias that might be a future biological limit to stable isotope-dose reduction in experiments on iron metabolism in man. Received: 9 February 2001 / Revised: 21 March 2001 / Accepted: 23 March 2001     

Enhancement of mass spectrometry performance for proteomic analyses using highfield asymmetric waveform ion mobility spectrometry (FAIMS)

Remarkable advances in mass spectrometry sensitivity and resolution have been accomplished over the past two decades to enhance the depth and coverage of proteome analyses. As these technological developments expanded the detection capability of mass spectrometers, they also revealed an increasing complexity of low abundance peptides, solvent clusters and sample contaminants that can confound protein identification. Separation techniques that are complementary and can be used in combination with liquid chromatography are often sought to improve mass spectrometry sensitivity for proteomics applications. In this context, high‐field asymmetric waveform ion mobility spectrometry (FAIMS), a form of ion mobility that exploits ion separation at low and high electric fields, has shown significant advantages by focusing and separating multiply charged peptide ions from singly charged interferences. This paper examines the analytical benefits of FAIMS in proteomics to separate co‐eluting peptide isomers and to enhance peptide detection and quantitative measurements of protein digests via native peptides (label‐free) or isotopically labeled peptides from metabolic labeling or chemical tagging experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Tracking Pathogen Infections by Time-Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time-resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host-cell proteins at different time points initiated by UV for label-free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella-containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

复杂生物体系中蛋白质高效分离分析技术的新进展

继人类基因组计划完成之后,作为一种新的研究策略,蛋白质组学在生命科学研究中发挥着愈来愈重要的作用。由于生物体系的复杂性和多样性,使得分离效率高、灵敏度高、通量高和动态范围宽的分离分析技术平台的研究和应用已成为蛋白质组学研究的重点和热点之一。着重介绍了近年来应用日益广泛的多维色谱预分离、毛细管液相色谱-质谱联用、毛细管电泳及其与质谱联用等高效分离分析技术在复杂生物体系的蛋白质分析中的最新进展。引用相关文献40篇。     

Tracking Pathogen Infections by Time‐Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time‐resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host‐cell proteins at different time points initiated by UV for label‐free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella‐containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

The potential of inorganic mass spectrometry in mineral and trace element nutrition research

Over the past two decades, new applications of inorganic mass spectrometry have been made possible by the use of stable isotopes as tracers in studies of mineral and trace element metabolism in man. Stable isotope techniques and radioisotope methods are the only reliable tools available for determination of the absorption, retention, or utilization of a nutrient by the human body. Recent developments in inorganic mass spectrometry might open new perspectives as progress in this field of research depends mainly on improving existing stable isotope techniques and on developing novel concepts. By improving precision in isotope analysis, isotope doses in experiments on man can be reduced to physiologically more meaningful levels. This will also enable reduction of the (often substantial) costs of isotopically labeling a nutrient in a test meal. Improvements in the mass spectrometric sensitivity will enable the development of new tracer techniques that have the potential to provide the information required by: 1. governmental institutions for designing food fortification programs; 2. the food industry for developing nutrient-fortified food products; and 3. public health authorities for establishing reliable dietary recommendations for intake of inorganic nutrients. In this context the current scope and limitations of thermal ionization mass spectrometry, inductively coupled mass spectrometry, accelerator mass spectrometry, and resonance ionization mass spectrometry are evaluated. Iron isotopic variations in the human body are discussed as a possible source of bias that might be a future biological limit to stable isotope-dose reduction in experiments on iron metabolism in man.     

Proteomic Studies of Syk-Interacting Proteins Using a Novel Amine-Specific Isotope Tag and GFP Nanotrap

Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein–protein interactions using anti-GFP single-chain antibody V_HH in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V_HH (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.     

A novel pyrimidine-based stable-isotope labeling reagent and its application to quantitative analysis using matrix-assisted laser desorption/ionization mass spectrometry

As an extension of our previous work, a novel pyrimidine-based stable-isotope labeling reagent, [d(0)]-/[d(6)]-4,6-dimethoxy-2-(methylsulfonyl)pyrimidine (DMMSP), was developed for comparative quantification of proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our one-step labeling strategy combines several desirable properties such as cysteine-specific labeling, signal amplification and direct analysis with minimum sample handling. All these features not only allow easy interpretation for protein identification and quantification but also ensure rapid and sensitive progression to MS analysis. Using cysteine, Cys-containing peptide, and lysozyme digest as model samples, the labeling methodology was established and the following pilot application for quantitative analysis was accomplished with high confidence, accuracy, efficiency, and reproducibility. The application of DMMSP-labeling strategy is expected to provide a powerful new tool for comparative proteome research, especially for the analysis of low-abundance proteins.