A prostate cancer tissue specific spectral library for targeted proteomic analysis

Prostate cancer is the most common cancer in males worldwide. Mass spectrometry-based targeted proteomics has demonstrated great potential in quantifying proteins from formalin-fixed paraffin-embedded (FFPE) and (fresh) frozen biopsy tissues. Here we provide a comprehensive tissue-specific spectral library for targeted proteomic analysis of prostate tissue samples. Benign and malignant FFPE prostate tissue samples were processed into peptide samples by pressure cycling technology (PCT)-assisted sample preparation, and fractionated with high-pH reversed phase liquid chromatography (RPLC). Based on data-dependent acquisition (DDA) MS analysis using a TripleTOF 6600, we built a library containing 108,533 precursors, 84,198 peptides and 9384 unique proteins (1% FDR). The applicability of the library was demonstrated in prostate specimens.     

Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.     

Identification and characterization of sulfolobus solfataricus P2 proteome using multidimensional liquid phase protein separations

We have identified and characterized the proteome of Sulfolobus solfataricus P2 using multidimensional liquid phase protein separations. Multidimensional liquid phase chromatography was performed using ion exchange chromatography in the first dimension, followed by reverse-phase chromatography using 500 microm i.d. poly(styrene-divinylbenzene) monoliths in the second dimension to separate soluble protein lysates from S. solfataricus. The 2DLC protein separations from S. solfataricus protein lysates enabled the generation of a 2D liquid phase map analogous to the traditional 2DE map. Following separation of the proteins in the second dimension, fractions were collected, digested in solution using trypsin and analyzed using mass spectrometry. These approaches offer significant reductions in labor intensity and the overall time taken to analyze the proteome in comparison to 2DE, taking advantage of automation and fraction collection associated with this approach. Furthermore, following proteomic analysis using 2DLC, the data obtained was compared to previous 2DE and shotgun proteomic studies of a soluble protein lysate from S. solfataricus. In comparison to 2DE, the results show an overall increase in proteome coverage. Moreover, 2DLC showed increased coverage of a number of protein subsets including acidic, basic, low abundance and small molecular weight proteins in comparison to 2DE. In comparison to shotgun studies, an increase in proteome coverage was also observed. Furthermore, 187 unique proteins were identified using 2DLC, demonstrating this methodology as an alternative approach for proteomic studies or in combination with 2DE and shotgun workflows for global proteomics.     

Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis

A high-throughput on-line capillary array-based two-dimensional liquid chromatography (2D-LC) system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallel capillary reversed-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumns. After salt fractionation, by directing identically split solvent-gradient flows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously with 18 capillary RP columns. LC effluents were directly deposited onto the MALDI target plates through an array of capillary tips at a 15-s interval, and then alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequent MALDI experiments. This new system allows an 18-fold increase in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by the analysis of a tryptic digest of proteins extracted from normal human liver tissue. A total of 462 proteins was identified, which proved the system's promising potential for high-throughput analysis and application in proteomics.     

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2‐D peptide separations employing mixed mode reversed phase anion exchange (MM (RP‐AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP‐AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP‐AX) compared with the traditional SCX hyphenated to RP‐LC‐MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP‐AX) for 2‐D LC proteomic studies was also extended in the analysis of iTRAQ‐labelled HeLa and cyanobacterial proteomes using nano‐flow chromatography interfaced to the MS/MS. We demonstrate MM (RP‐AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.     

评价新型稳定同位素赖氨酸标记在定量蛋白质组学中的应用

为了评价基于2-甲氧基-4,5-二氢-1氢-咪唑稳定同位素试剂在定量蛋白质组学中的应用价值,合成了轻型(D0)和重型(D4)的2-甲氧基-4,5-二氢-1氢-咪唑,通过对标准蛋白BSA酶解后产物的标记确认标记反应的特异性,并观察了标记物在MALDI-TOF-MS和LC-ESI-MS中定量的准确性,标记肽在串联质谱中的离子特点,以及对反相液相色谱行为的影响。结果表明,2-甲氧基-4,5-二氢-1氢-咪唑只与酶解后的肽段赖氨酸侧链氨基反应,具有良好的标记特异性;差异表达蛋白的定量可以通过MALDI和ESI电离模式实现;标记肽的串联质谱主要产生y离子,测序更为简便;反相液相色谱可以保持较好的分离效果,氘原子的引入不会影响保留时间,侧链修饰可以用于涉及液相色谱分离的蛋白质组学技术。2-甲氧基-4,5-二氢-1氢-咪唑稳定同位素试剂可以用于定量蛋白质组学。     

Comparative study of [Three] LC-MALDI workflows for the analysis of complex proteomic samples

Large-scale proteomic analyses frequently rely on high-resolution peptide separation of digested protein mixtures in multiple dimensions to achieve accuracy in sample detection and sensitivity in dynamic range of coverage. This study was undertaken to demonstrate the feasibility of MALDI MS/MS with off-line coupling to HPLC for the analysis of whole cell lysates of wild-type yeast by three different workflows: SCX-RPHPLC-MS/MS, high-pH SAX-RPHPLC-MS/MS and RP (protein)-SCX-RPHPLC-MS/MS. The purpose of these experiments was to demonstrate the effect of a workflow on the end results in terms of the number of proteins detected, the average peptide coverage of proteins, and the number of redundant peptide sequencing attempts. Using 60 microg of yeast lysate, minor differences were seen in the number of proteins detected by each method (800-1200). The most significant differences were observed in redundancy of MS/MS acquisitions.     

Highly sensitive proteome analysis of FACS-sorted adult colon stem cells

In proteomics, multidimensional liquid chromatography combined with mass spectrometry has become a standard technique to reduce sample complexity and tackle the vast dynamic range. Such fractionation is necessary to obtain a comprehensive analysis of biological samples such as tissues and cell lines. However, extensive fractionation comes at the expense of sample losses, hampering the analysis of limited material. We previously described a highly sensitive multidimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography and reversed phase chromatography, which allows proteomic analysis with minimal sample losses. Here we apply this strategy to the analysis of a limited number of FACS-sorted colon stem cells extracted from mouse intestine, obtaining a proteome coverage comparable to current methods that generally require 100-fold more starting material. We propose that this alternative multidimensional chromatographic technology will find ample application such as in the analysis of distinct cellular populations obtained by laser microdissection.     

Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.     

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.     

Two‐step OFFGEL approach for effective peptide separation compatible with iTRAQ labeling

Shotgun proteomic analyses are increasingly becoming methods of choice for complex samples. The development of effective methods for fractionating peptides to reduce the complexity of the sample before mass analysis is a key point in this strategy. The OFFGEL technology has recently become a tool of choice in proteomic analysis at peptide level. This OFFGEL electrophoresis (OGE) approach allows the in‐solution separation of peptides from various biological sources by isoelectric focusing in highly resolved 24 fractions. It was also demonstrated that OGE technology is a filtering tool for pI‐based validation of peptide identification. As peptide OGE is compatible with iTRAQ labeling, OGE is finding valuable applications in quantitative proteomics as well. The aim of this study is to explain a new 2D‐OGE approach that improves the proteomic coverage of complex mixtures such as colorectal cell line lysates, and which is compatible with iTRAQ labeling.     

Microchip reactor packed with metal-ion chelated magnetic silica microspheres for highly efficient proteolysis

An easily replaceable and regenerable protease microreactor with metal-ion chelated adsorption of enzyme has been fabricated on chip. Magnetic microspheres with small size (approximately 200 nm in diameter) and strong magnetism were synthesized and were modified with tetraethyl orthosilicate. The metal chelating agent of iminodiacetic acid was then reacted with glycidoxypropyltrimethoxysilane before its immobilization onto the surface of magnetic silica microspheres (MS microspheres). The metal ion of copper and enzyme were subsequently adsorbed onto the surface. The prepared MS microspheres were then locally packed into the microchannel by the application of a strong magnetic field using a magnet to form an on-chip enzymatic microreactor. Capability of the proteolytic microreactor was demonstrated by cytochrome c and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF/TOF MS with sequence coverage of 77% and 21% observed, respectively. This microreactor was also applied to the analysis of one RPLC fraction of rat liver extract. After a database search, 23 unique peptides corresponding to 7 proteins were identified when one RPLC fraction of rat liver extract was digested by the microreactor. This opens a route for its future application in top-down proteomic analysis.     

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.     

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.     

Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines

An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research.     

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.     

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.     

Multidimensional protein profiling technology and its application to human plasma proteome

In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.     

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 ~10³) 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 10⁶ 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.