Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics

Recent developments in combined separations with mass spectrometry for sensitive and high-throughput proteomic analyses are reviewed herein. These developments primarily involve high-efficiency (separation peak capacities of approximately 10(3)) nanoscale liquid chromatography (flow rates extending down to approximately 20 nl/min at optimal liquid mobile-phase separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry and in particular, high-sensitivity and high-resolution Fourier transform ion cyclotron resonance mass spectrometry. Such approaches enable analysis of low nanogram level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole level. The resultant protein measurement dynamic range can approach 10(6) for nanogram-sized proteomic samples, while more abundant proteins can be detected from subpicogram-sized (total) proteome samples. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high-quality routine performance nanoscale proteomic analyses is also discussed.     

生物质谱与蛋白质组学

蛋白质组学是后基因组学时代最受关注的研究领域之一,其核心的鉴定技术——生物质谱近年来在仪器设计以及鉴定通量、分辨率和灵敏度等各方面均有质的飞跃,促进了蛋白质表达谱作图、定量蛋白质组分析、亚细胞器蛋白质组作图、蛋白质翻译后修饰以及蛋白质相互作用等蛋白质组研究各个领域的飞速发展。本综述了生物质谱技术的最新进展,及其在蛋白质组学研究中的应用。     

Surface plasmon resonance mass spectrometry in proteomics

Due to the enormous complexity of the proteome, focus in proteomics shifts more and more from the study of the complete proteome to the targeted analysis of part of the proteome. The isolation of this specific part of the proteome generally includes an affinity-based enrichment. Surface plasmon resonance (SPR), a label-free technique able to follow enrichment in real-time and in a semiquantitative manner, is an emerging tool for targeted affinity enrichment. Furthermore, in combination with mass spectrometry (MS), SPR can be used to both selectively enrich for and identify proteins from a complex sample. Here we illustrate the use of SPR-MS to solve proteomics-based research questions, describing applications that use very different types of immobilized components: such as small (drug or messenger) molecules, peptides, DNA and proteins. We evaluate the current possibilities and limitations and discuss the future developments of the SPR-MS technique.     

Trends in mass spectrometry instrumentation for proteomics

Mass spectrometry has become a primary tool for proteomics because of its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the need for increased capabilities for proteome measurements is immense and is now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements and promise more than order of magnitude improvements in sensitivity, dynamic range and throughput for proteomic analyses in the near future.     

High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behavior in strong and weak electric fields. FAIMS is easily interfaced with electrospray ionization and has been implemented as an additional separation mode between liquid chromatography (LC) and mass spectrometry (MS) in proteomic studies. FAIMS separation is orthogonal to both LC and MS and is used as a means of on-line fractionation to improve the detection of peptides in complex samples. FAIMS improves dynamic range and concomitantly the detection limits of ions by filtering out chemical noise. FAIMS can also be used to remove interfering ion species and to select peptide charge states optimal for identification by tandem MS. Here, the authors review recent developments in LC-FAIMS-MS and its application to MS-based proteomics.     

Mass spectrometry technologies for proteomics.

In the late 1980s, the advent of soft ionization techniques capable of generating stable gas phase ions from thermally unstable biomolecules, namely matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), laid the way for the development of a set of powerful alternatives to the traditional Edman chemistry for the structural characterization of peptides and proteins. The rapid protein identification capabilities that, coupled with two-dimensional gel electrophoresis, provided insights into all sorts of biological systems since the dawn of proteomics and have been exploited in the last few years for the development of more powerful and automatable gel-free strategies, mainly based on multidimensional chromatographic separations of peptides from proteolytic digests. In parallel to the evolution of ion sources, mass analysers and scan modes, the invention of new elegant biochemical strategies to fractionate or simplify highly complex mixtures, or to introduce isotopic labels in peptides in a variety of ways now makes also possible large-scale, high-coverage quantitative studies in a wide dynamic range. In this review, we provide the fundamental concepts of mass spectrometry (MS) and describe the technological progress of MS-based proteomics since its earliest days. Representative literature examples of their true power, either when employed as exploratory or as targeted techniques, is provided as well.     

Bioinformatics in mass spectrometry data analysis for proteomics studies

Mass spectrometry is a technique widely employed for the identification and characterization of proteins. The role of bioinformatics is fundamental for the elaboration of mass spectrometry data due to the amount of data that this technique can produce. To process data efficiently, new software packages and algorithms are continuously being developed to improve protein identification and characterization in terms of high-throughput and statistical accuracy. However, many limitations exist concerning bioinformatics spectral data elaboration. This review aims to critically cover the recent and future developments of new bioinformatics approaches in mass spectrometry data analysis for proteomics studies.     

Applications of MALDI-TOF mass spectrometry in clinical proteomics

Introduction: The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics.

Areas covered: This review aims to provide an updated overview of MALDI-TOF MS applications in clinical proteomics. The most relevant features of this analysis have been discussed, highlighting both pre-analytical and analytical factors that are crucial in proteomics studies. Particular emphasis is placed on biofluids proteomics for biomarkers discovery and on recent progresses in clinical microbiology, drug monitoring, and minimal residual disease (MRD).

Expert commentary: Despite some analytical limitations, the latest technological advances together with the easiness of use, the low time and low cost consuming and the high throughput are making MALDI-TOF MS instruments very attractive for the clinical practice. These features offer a significant potential for the routine of the clinical laboratory and ultimately for personalized medicine.     

Target class strategies in mass spectrometry-based proteomics

The cornerstone of proteomics resides in using traditional methods of protein chemistry, to extract and resolve complex mixtures, in concert with the powerful engines of mass spectrometry to decipher peptide and protein identities. The broad utility of proteomics technologies to map protein interactions, understand regulatory mechanisms and identify biomarkers associated with disease states and drug treatments necessitates a targeted biochemical approach tailored to the characteristics of the tissue, fluid or cellular extract being studied. The application of affinity methods in proteomic studies to focus on particular classes of molecules is being used with increasing frequency and comprises the subject of this review. An overview of successfully applied affinity methods is provided, along with speculation on the use of innovative approaches. Sample preparation and processing are critical for proteomics with affinity reagents, as only functional and active proteins can be isolated in most cases. Considerations for methods of sample preparation to optimize affinity capture and release are also discussed.     

Multidimensional peptide separations in proteomics

Multidimensional peptide separation will play an increasingly important role in the drive to identify and quantitate the proteome. By increasing the peak and load capacity, multidimensional approaches increase the number and dynamic range of peptides that can be analyzed in a complex biological organism. Separation methods using different physical properties of peptides have been combined with varying degrees of success. The ultimate goal is a rapid separation strategy that can be coupled with analytical methods, such as mass spectrometry, to provide comprehensive monitoring of the changing concentration, interactions, and structures of proteins in the proteome.     

The discovery of protein biomarkers in pre-eclampsia: the promising role of mass spectrometry

Abstract

Context: Pre-eclampsia (PE) is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. The aetiology of the disease remains poorly understood with lack of reliable diagnostic tests. PE is a multisystem disorder so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. Mass spectrometry (MS) is indispensable analytical tool in protein analysis studies. MS-based proteomics have the ability to detect the entire protein complement to provide a useful window into a range of biological processes and allow the identification of differentially expressed proteins between samples.

Objective: The aim of this review is to summarise, discuss and evaluate the current predominant MS-based approaches applied for protein biomarker discovery. The paper also seeks to evaluate the current potential PE biomarkers described in the literature and identify issues that can guide future research.

Conclusion: MS-based proteomics studies are promising alternatives to classical hypothesis-driven approaches to discover novel biomarkers and provide new insights into the underlying phathophysiological mechanisms of PE. This should aid in the early diagnosis of PE and the understanding of the aetiology of the disease.     

基于质谱技术的蛋白质组定量方法

对不同状态下的蛋白质在表达和修饰水平上进行精确定量,对于探索蛋白质的生物功能、发现疾病的生物标志物都具有重要意义,也是当前蛋白质组学的一个重要研究前沿。近年来,各种蛋白质组定量的新技术和新方法不断涌现,但仍面临着巨大挑战。本文就基于质谱技术的多种蛋白质组定量方法的基本原理、近几年的研究进展和应用进行评述。     

Absolute quantification strategies in proteomics based on mass spectrometry

The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.     

基于电子捕获裂解/电子转运裂解串联质谱技术的蛋白质组学研究

蛋白质组学的兴起带动了质谱技术的快速发展,而质谱技术的进步则拓宽了蛋白质组学研究问题的广度.最近10年内,肽段或完整蛋白质在质谱仪中的裂解技术——电子捕获裂解(electron capture dissociation,ECD)与电子转运裂解(electron transfer dissociation,ETD)逐渐发展起来.ECD和ETD在蛋白质组学中的应用,特别是在蛋白质的翻译后修饰鉴定和自顶而下(Top-down)的完整蛋白质裂解研究中已经展示出了诱人的前景.对ECD和ETD的基本原理、质谱特点、仪器实现、数据解析算法与软件开发,以及在蛋白质组学中的应用进展等方面进行了比较系统全面的阐述,并对当前的研究问题、面临的技术挑战与未来的发展趋势等方面作了深入剖析.     

Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome.     

High-throughput proteomics using Fourier transform ion cyclotron resonance mass spectrometry

The advent of high-throughput proteomic technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of the cellular machinery. Here, recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry are reviewed along with its potential application to high-throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system level.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Mass spectrometry is only one piece of the puzzle in clinical proteomics.

Biomarker discovery in clinical proteomics is being performed on relatively large patient cohorts by utilizing the high throughput of laser desorption/ionization mass spectrometry (MALDI- and SELDI-TOF-MS). Dealing directly with patient samples as opposed to working in cell or animal systems requires a host of considerations both before and after mass spectrometric analysis to obtain robust biomarker candidates. The challenges associated with the heterogeneity of typical samples are amplified by the ability to detect hundreds to thousands of proteins simultaneously. Adherence to protocols and consistency, however, can ensure optimal results. A study starts necessarily with a relevant clinical question and proceeds to a planning phase where sample availability, statistical test selection, logistics and bias reduction are key points. The physical analysis requires consistency and standardized protocols that are helped significantly through automation. Data analysis is broken into two stages, screening and final testing, which can detect either single candidates or a pattern of proteins. Biomarker identification can be performed at this point and will help significantly in the last stage, interpretation. Replication should be performed in an independent sample set in a separate study. The candidate biomarkers from an initial study give a wealth of information that can help to pinpoint patient subpopulations for a more exhaustive proteomic study using complementary platforms with limited capacity but extremely high information content. A clinical proteomics pilot project can also lead to better selection of model systems by providing a direct link with patient samples.     

Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Antibody‐based microarrays is a rapidly evolving technology that has gone from the first proof‐of‐concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high‐ as well as low‐abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in‐depth global proteome surveys, we propose to interface affinity proteomics with MS‐based read‐out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5–100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10⁴ individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif‐specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif‐containing peptides are specifically captured, enriched and subsequently detected and identified using MS.     

A streamlined approach to high-throughput proteomics

Proteomics has rapidly become an important tool for life science research, allowing the integrated analysis of global protein expression from a single experiment. To accommodate the complexity and dynamic nature of any proteome, researchers must use a combination of disparate protein biochemistry techniques, often a highly involved and time-consuming process. Whilst highly sophisticated, individual technologies for each step in studying a proteome are available, true high-throughput proteomics that provides a high degree of reproducibility and sensitivity has been difficult to achieve. The development of high-throughput proteomic platforms, encompassing all aspects of proteome analysis and integrated with genomics and bioinformatics technology, therefore represents a crucial step for the advancement of proteomics research. ProteomIQ? (Proteome Systems) is the first fully integrated, start-to-finish proteomics platform to enter the market. Sample preparation and tracking, centralized data acquisition and instrument control, and direct interfacing with genomics and bioinformatics databases are combined into a single suite of integrated hardware and software tools, facilitating high reproducibility and rapid turnaround times. This review will highlight some features of ProteomIQ, with particular emphasis on the analysis of proteins separated by 2D polyacrylamide gel electrophoresis.