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.     

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.     

The utility of proteases in proteomics,from sequence profiling to structure and function analysis

In mass spectrometry (MS)-based bottom-up proteomics, protease digestion plays an essential role in profiling both proteome sequences and post-translational modifications (PTMs). Trypsin is the gold standard in digesting intact proteins into small-size peptides, which are more suitable for high-performance liquid chromatography (HPLC) separation and tandem MS (MS/MS) characterization. However, protein sequences lacking Lys and Arg cannot be cleaved by trypsin and may be missed in conventional proteomic analysis. Proteases with cleavage sites complementary to trypsin are widely applied in proteomic analysis to greatly improve the coverage of proteome sequences and PTM sites. In this review, we survey the common and newly emerging proteases used in proteomics analysis mainly in the last 5 years, focusing on their unique cleavage features and specific proteomics applications such as missing protein characterization, new PTM discovery, and de novo sequencing. In addition, we summarize the applications of proteases in structural proteomics and protein function analysis in recent years. Finally, we discuss the future development directions of new proteases and applications in proteomics.     

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.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.     

The Drosophila melanogaster sperm proteome-II (DmSP-II)

Advances in mass spectrometry technology, high-throughput proteomics and genome annotations have resulted in significant increases in our molecular understanding of sperm composition. Using improved separation and detection methods and an updated genome annotation, a re-analysis of the Drosophila melanogaster sperm proteome (DmSP) has resulted in the identification of 956 sperm proteins. Comparative analysis with our previous proteomic dataset revealed 766 new proteins and an updated sperm proteome containing a total of 1108 proteins, termed the DmSP-II. This expanded dataset includes additional proteins with predicted sperm functions and confirms previous findings concerning the genomic organization of sperm loci. Bioinformatic and protein network analyses demonstrated high quality and reproducibility of proteome coverage across three replicate mass spectrometry runs. The use of whole-cell proteomics in conjunction with characterized phenotypes, functional annotations and pathway information has advanced our systems level understanding of sperm proteome functional networks.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein–protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.     

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.     

Proteomic technologies in modern biomedical science

This review highlights modern technologies employed in proteomics. Methods of sample preparations are discussed with special emphasis on the requirements for preparation of biological material, which may seriously influence the results of proteomic studies. Methods of solubilization, electrophoresis, chromatographic protein separation, and visualization of protein spots in gels are described. Modern methods of mass spectrometry used in proteomic studies include combination of protein chips with mass spectrometry. The review also describes approaches of functional proteomics, i.e., interactomics, and also bioinformatic resources used in proteomics for image analysis of 2D-gel-electrophoresis and for identification of protein sequences by mass spectra.     

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.     

蛋白质组学研究中的分离分析技术及其研究进展

在后基因组时代,蛋白质组学成为新的研究热点。蛋白质组学的研究目标是为复杂蛋白质样品建立一个高通量、大规模、自动化的分离分析技术平台,从而实现准确、快速地筛选功能蛋白质。蛋白质的分离分析在蛋白组学研究中起着非常重要的作用。本文主要综述在蛋白质组学研究中二维凝胶电泳、毛细管电泳及其与质谱联用、多维液相分离技术及其与质谱联用和蛋白质芯片等高效分离分析技术的应用研究进展。     

类芽孢杆菌(Paenibacillus sp.)蛋白质组学研究中三种蛋白提取方法的比较分析

【目的】革兰氏阳性类芽孢杆菌（Paenibacillus sp.）本身细胞壁的结构特点导致其菌体全蛋白不易获得。本研究选取了3种破碎方法——溶菌酶联合超声破碎法（方法一）、溶菌酶联合SDS热处理破碎法（方法二）、液氮联合超声破碎法（方法三）进行革兰氏阳性菌的细胞破碎,以期获得适于样品菌株基于质谱技术进行蛋白质组学研究的制备方法。【方法】在蛋白样品的制备过程中,对3种不同破碎方法的蛋白提取得率和SDS-PAGE检测分析结果进行比较;随后将3种蛋白样品制备方法的样品用质谱技术进行鉴定,分析不同蛋白样品基于质谱技术鉴定蛋白的差异。【结果】在蛋白样品的制备提取过程中,不同破碎方法的蛋白提取率大致相同。用单因素方差比较3种提取方法质谱鉴定蛋白数的差异性,方法三鉴定的蛋白数最多（2 638个）,其次是方法一（2 452个）,方法二鉴定的蛋白数最少（2 003个）。进一步用韦恩图分析比较不同提取方法的蛋白鉴定通量差异,综合考虑蛋白提取效率的结果以及液氮研磨法提取蛋白的缺点,最终选取溶菌酶联合超声破碎法（方法一）提取菌株全蛋白作为该菌基于质谱分析其蛋白质组学研究中最适合的方法。最后,对质谱鉴定菌株蛋白包括分子量、等电点、疏水性的基本性质进行分析,发现3种破碎方法质谱鉴定的蛋白与模式菌株多黏类芽孢杆菌（Paenibacillus polymyxa）基因组中预测蛋白的各个组分分布占比基本一致,都保证了菌株蛋白质组数据信息的完整性。【结论】基于质谱技术开展革兰氏阳性类芽孢杆菌（Paenibacillus sp.）的蛋白质组学研究,溶菌酶联合超声破碎法是提取该菌株全蛋白最适合的方法。     

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.^1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.^4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.⁶ Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.^7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.^10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.     

Alternative profiling platform based on MELDI and its applicability in clinical proteomics

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.     

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.     

Protein recovery and identification from the gulf killifish, Fundulus grandis: comparing snap-frozen and RNAlater® preserved tissues

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one‐dimensional (1‐D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap‐freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater^®. In liver and heart, the protein profiles in 1‐D gels were better preserved by snap‐freezing, while in gill, the 1‐D protein profile was better preserved by immersion in RNAlater^®. In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC‐MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.     

Optimization of 2D-PAGE protocols for proteomic analysis of two nonaxenic toxin-producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp.

Aims:  To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
Methods and Results:  The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4–7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS).
Conclusions:  The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp.
Significance and Impact of the Study:  We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.     

Separation methodology to improve proteome coverage depth

So-called ‘in-depth proteomics’ and its applied separation methodology to improve the proteome coverage depth has become an important issue in mass spectrometric-based proteomics and system-wide cell biology studies. Employing a bottom-up approach and a variety of separation techniques, it allows for identification of proteins with low copy numbers and enables researchers to correlate the number of expressed genes in a cell with the proteome. Here we describe recent advances in this field with emphasis on peptide and protein separation technologies. The discussion is focused both on single injection analyses employing long reversed phase liquid chromatography separations of peptides (‘single shot proteomics’) and on the combination of orthogonal protein and peptide separation methods to achieve maximum protein coverage. Owing to these improvements, in-depth proteomics has now fully entered the field and is being implemented in an increasing number of laboratories.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.