The human plasma proteome: analysis of Chinese serum using shotgun strategy

We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20 healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1) removal of six high-abundant proteins; (2) tryptic digestion of low- and high-abundant proteins of serum; (3) separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944 nonredundant proteins were identified under a stringent filter condition (X(corr) > or = 1.9, > or = 2.2, and > or = 3.75, < or = C(n) > or = 0.1, and R(sp) > or = 4.0) in both pooled male and female samples, in which 594 and 622 entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206 proteins were identified with at least two distinct peptides per protein and 185 proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.     

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

The core prerequisites for an efficient proteome-scale analysis of mammalian membrane proteins are effective isolation, solubilization, digestion and multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). This protocol is for analysis of the mammalian membrane proteome that relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol. Tryptic digestion is followed by strong cation exchange (SCX) chromatography and reversed phase (RP) chromatography coupled online with MS/MS for protein identification. The use of a methanol-based buffer eliminates the need for reagents that interfere with chromatographic resolution and ionization of the peptides (e.g., detergents, chaotropes, inorganic salts). Sample losses are minimized because solubilization and digestion are carried out in a single tube avoiding any sample transfer or buffer exchange between these steps. This protocol is compatible with stable isotope labeling at the protein and peptide level, enabling identification and quantitation of integral membrane proteins. The entire procedure--beginning with isolated membrane fraction and finishing with MS data acquisition--takes 4-5 d.     

Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis

With the completion of the sequencing of the Arabidopsis genome and with the significant increase in the amount of other plant genome and expressed sequence tags (ESTs) data, plant proteomics is rapidly becoming a very active field. We have pursued a high-throughput mass spectrometry-based proteomics approach to identify and characterize membrane proteins localized to the Arabidopsis thaliana chloroplastic envelope membrane. In this study, chloroplasts were prepared from plate- or soil-grown Arabidopsis plants using a novel isolation procedure, and "mixed" envelopes were subsequently isolated using sucrose step gradients. We applied two alternative methodologies, off-line multidimensional protein identification technology (Off-line MUDPIT) and one-dimensional (1D) gel electrophoresis followed by proteolytic digestion and liquid chromatography coupled with tandem mass spectrometry (Gel-C-MS/MS), to identify envelope membrane proteins. This proteomic study enabled us to identify 392 nonredundant proteins.     

Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in <10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

Application of displacement chromatography for the analysis of a lipid raft proteome

Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC–MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC–MS approach.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas

Human blood plasma can be obtained relatively noninvasively and contains proteins from most, if not all, tissues of the body. Therefore, an extensive, quantitative catalog of plasma proteins is an important starting point for the discovery of disease biomarkers. In 2005, we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of proteins, and that a comprehensive plasma proteome can be compiled only by combining data from many different experiments. Applying advanced computational methods developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptides, we have now compiled a high-confidence human plasma proteome reference set with well over twice the identified proteins of previous high-confidence sets. It includes a hierarchy of protein identifications at different levels of redundancy following a clearly defined scheme, which we propose as a standard that can be applied to any proteomics data set to facilitate cross-proteome analyses. Further, to aid in development of blood-based diagnostics using techniques such as selected reaction monitoring, we provide a rough estimate of protein concentrations using spectral counting. We identified 20,433 distinct peptides, from which we inferred a highly nonredundant set of 1929 protein sequences at a false discovery rate of 1%. We have made this resource available via PeptideAtlas, a large, multiorganism, publicly accessible compendium of peptides identified in tandem MS experiments conducted by laboratories around the world.     

Addressing trypsin bias in large scale (phospho)proteome analysis by size exclusion chromatography and secondary digestion of large post-trypsin peptides

In the vast majority of bottom-up proteomics studies, protein digestion is performed using only mammalian trypsin. Although it is clearly the best enzyme available, the sole use of trypsin rarely leads to complete sequence coverage, even for abundant proteins. It is commonly assumed that this is because many tryptic peptides are either too short or too long to be identified by RPLC-MS/MS. We show through in silico analysis that 20-30% of the total sequence of three proteomes (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Homo sapiens) is expected to be covered by Large post-Trypsin Peptides (LpTPs) with M(r) above 3000 Da. We then established size exclusion chromatography to fractionate complex yeast tryptic digests into pools of peptides based on size. We found that secondary digestion of LpTPs followed by LC-MS/MS analysis leads to a significant increase in identified proteins and a 32-50% relative increase in average sequence coverage compared to trypsin digestion alone. Application of the developed strategy to analyze the phosphoproteomes of S. pombe and of a human cell line identified a significant fraction of novel phosphosites. Overall our data indicate that specific targeting of LpTPs can complement standard bottom-up workflows to reveal a largely neglected portion of the proteome.     

Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics.

Shotgun proteomics, where a tryptic digest of a complex proteome sample is directly analyzed by either single dimensional or multidimensional liquid chromatography tandem mass spectrometry, has gained acceptance in the proteomics community at large and is widely used in core facilities. Here we review the development in our laboratory of an alternative first-dimension separation technique for shotgun proteomics, immobilized pH gradient isoelectric focusing (IPG-IEF). The key advantages of the technology over other multidimensional separation formats (simplicity, high resolution, and high sensitivity) are discussed. The concept of using peptide pI to filter large shotgun proteomics datasets generated by the IPG-IEF technique to minimize false positives and negatives is also introduced. Finally, an account of the comparison of the technique with the established gold standard for multidimensional separation of peptides, strong cation exchange chromatography, is presented, along with the prospects for the use of peptide pI along with accurate mass measurement for the identification of peptides.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

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.     

Preparation and application of a partially degradable gel in mass spectrometry-based proteomic analysis

In-gel digestion is an attractive route in mass spectrometry-based proteomic analysis, which, however, often suffers from a certain amount of sample loss mainly due to insufficient protein digestion and peptide extraction. To address this, herein we establish a partially degradable gel-assisted protein digestion and peptide recovery method by means of a simple replacement of bis-acrylamide (BA) with bis-acrylylcystamine (BAC). Concretely, the protein sample solubilized using high concentrations of sodium dodecyl sulfate (SDS) and urea were directly entrapped and immobilized into BAC-crosslinked gel by vacuum-dried gel absorption followed by fixation treatment. After removal of SDS and urea by repeated washing, the proteins were subjected to in-gel digestion and the gel was reductively treated. The tryptic peptides were recovered from the partial degradation of the gel and analyzed afterwards by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). Compared with conventional BA-crosslinked gel method, this new method increased the numbers of identified proteins and unique peptides by 20.2% and 20.4%, respectively. The further statistical analysis demonstrated that the method improved the recovery of tryptic peptides particularly larger and/or hydrophobic peptides, thereby significantly facilitating protein identification. Thus, the newly developed method is a promising alternative for BA-crosslinked gel-based shotgun workflows and has potential application in the related fields of protein chemistry and proteomics.     

Identification of oxidized proteins in rat plasma using avidin chromatography and tandem mass spectrometry

The objective in much of the proteomics literature today is to establish the difference between healthy and disease states at the protein level using blood plasma. A critical component in this endeavor is to establish what is normal. The focus of the work reported here was to do this with oxidized proteins that might relate to oxidative stress and oxidative stress-related diseases. Oxidative stress is known to increase markedly in cancer, diabetes, heart disease, and neurodegenerative diseases. Since proteins are one of the targets of ROS, generated by oxidative stress, oxidized proteins are excellent biomarker candidates for these diseases. But first it is necessary to identify oxidized proteins that occur in the healthy state. Healthy rat plasma was used in this study as a source for the identification of naturally oxidized proteins. Freshly drawn blood was treated with biotin hydrazide to selectively derivatize carbonyl groups in oxidized proteins. Oxidized proteins thus biotinylated were separated from the other plasma proteins using avidin affinity chromatography. Affinity selected proteins were further fractionated on a C(8) RP column and fractions collected. The collected fractions were then tryptic digested and the peptides identified using a combination of LC/MS/MS and database searches. One hundred forty-six proteins were identified using 700 signature peptides from the tryptic digested chromatographic fractions. The most frequently encountered proteins in the samples were keratins. Brain and liver were among the organs contributing the most oxidized proteins to plasma followed by heart and kidney.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Proteomic analysis of Bacillus anthracis Sterne vegetative cells

Mass spectrometry and proteomics have found increasing use as tools for the rapid detection of pathogenic bacteria, even when they are in a mixture of non-pathogenic relatives. The success of this technique is greatly augmented by the availability of publicly accessible proteomic databases for specific pathogenic bacteria. To aid proteomic detection analyses for the causative agent of anthrax, we have constructed a comprehensive proteomic catalogue of vegetative Bacillus anthracis Sterne cells using liquid chromatography tandem-mass spectrometry. Proteins were separated by molecular weight or isoelectric point prior to tryptic digestion. Alternatively, the whole protein extract was digested and tryptic peptides were separated by cation exchange chromatography prior to Reverse Phase-LC-MS/MS. The use of three complementary, pre-analytical separation techniques resulted in the identification of 1048 unique proteins, including 694 cytosolic, 153 membrane (including 27 cell wall), and 30 secreted proteins, accounting for 19% of the total predicted proteome. Each identified protein was functionally categorized using the gene attribute database from TIGR CMR. These results provide a large proteomic catalogue of vegetative B. anthracis cells and, coupled with the recent proteomic catalogue of B. anthracis spore proteins, form a thorough summary of proteins expressed in the active and dormant stages of this organism.     

Human plasma proteome analysis by reversed sequence database search and molecular weight correlation based on a bacterial proteome analysis

In shotgun proteomics, proteins can be fractionated by 1-D gel electrophoresis and digested into peptides, followed by liquid chromatography to separate the peptide mixture. Mass spectrometry generates hundreds of thousands of tandem mass spectra from these fractions, and proteins are identified by database searching. However, the search scores are usually not sufficient to distinguish the correct peptides. In this study, we propose a confident protein identification method for high-throughput analysis of human proteome. To build a filtering protocol in database search, we chose Pseudomonas putida KT2440 as a reference because this bacterial proteome contains fewer modifications and is simpler than the human proteome. First, the P. putida KT2440 proteome was filtered by reversed sequence database search and correlated by the molecular weight in 1-D-gel band positions. The characterization protocol was then applied to determine the criteria for clustering of the human plasma proteome into three different groups. This protein filtering method, based on bacterial proteome data analysis, represents a rapid way to generate higher confidence protein list of the human proteome, which includes some of heavily modified and cleaved proteins.     

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.