Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry

Two-dimensional liquid chromatography (2D-LC) coupled on-line with electrospray ionization tandem mass spectrometry (2D-LC-ESI-MS/MS) is a new platform for analysis and identification of proteome. Peptides are separated by 2D-LC and then performed MS/MS analysis by tandem MS/MS. The MS/MS data are searched against database for protein identification. In one 2D-LC-ESI-MS/MS run, we obtained not only the structural information of peptides directly from MS/MS, but also the retention time of peptides eluted from LC. Information on the chromatographic behavior of peptides can assist protein identification in the new platform for proteomics. The retention time of the matching peptides of the identified protein was predicted by the hydrophobic contribute of each amino acid on reversed-phase liquid chromatography (RPLC). By using this strategy proteins were identified by four types of information: peptide mass fingerprinting (PMF), sequence query, and MS/MS ions searched and the predicted retention time. This additional information obtained from LC could assist protein identification with no extra experimental cost.     

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.     

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.     

多维液相色谱分离技术在复杂蛋白质组学样品鉴定中的应用

目的：随着蛋白组学技术的发展,液相色谱-串联质谱的联用技术（液质联用）逐渐成为蛋白组学的主流技术。方法：通过结合各种不同原理的色谱分离类型,多维液相色谱分离技术能够极大的提高分离系统的峰容量,达到有效分离复杂程度很高的蛋白质组学样品的目的。结果：最广泛使用的多维液相色谱分离系统是离子交换色谱（IEX）和反相色谱（RP）的二维结合,近年来又发展出了分离能力更强的三维液相色谱分离系统,并且已经在蛋白质组学研究中得到了应用。结论：本文综述了多种多维液相色谱分离方法,在这些方法中,不同的分离原理的色谱类型被用于肽段或蛋白混合物的预分离中,有效促进了样品的充分分离,极大地提高了复杂样品的蛋白组学鉴定能力。     

Methods for on-chip protein analysis

The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed.     

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.     

牛血清白蛋白胰蛋白酶酶解产物的色谱-质谱联用分析及其三种数据库搜寻鉴定方法的比较

利用反相高效液相色谱 (RP HPLC)和电喷雾串联质谱 (ESI MS MS)联用技术直接对模式蛋白分子 (牛血清白蛋白 ,BSA)的胰蛋白酶酶解产物进行分离和测定 .获得的一系列BSA酶解片段的一级 (MS)和二级 (MS MS)质谱数据经分析软件处理后 ,分别在不同处理和不同参数条件下 ,用 3种不同的方法通过网上蛋白质数据库进行蛋白质搜寻鉴定 .结果显示 ,3种搜寻法都能正确地鉴定该蛋白质 ,其中以利用MS数据的肽质量指纹谱搜寻法 (PMF法 )较为快捷方便 ,但鉴定结果易受数据处理和数据库搜寻鉴定时参数设置等因素的影响 ;利用未解析MS MS数据 (rawMS MSdata)的搜寻法可在较宽的搜寻参数变化范围内获得明确的鉴定结果 ;而借助从头测序 (denovosequencing)结果的序列搜寻法 (sequencequery)则显示出更高的专一性 ,利用较少酶解片段数据就能得到稳定和明确的鉴定结果 ,搜寻参数变化的影响很小 .就酶解条件、数据处理和搜寻参数设置对蛋白质鉴定结果的影响展开详细的讨论 ,为蛋白质组学研究中的数据处理和库搜寻鉴定积累了可借鉴的资料     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

The human plasma proteome: a nonredundant list developed by combination of four separate sources

We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

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.     

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.     

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.     

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.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.     

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.     

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.     

Proteome profiling of the green sulfur bacterium Chlorobaculum tepidum by N-terminal proteomics

In this study, we performed the first large-scale identification of N-terminal peptides from the green sulfur bacterium Chlorobaculum tepidum. Combined fractional diagonal chromatography (COFRADIC) was used to isolate protein N-terminal peptides from three different proteome preparations, and following LC-MS/MS analysis, over 621 different proteins were identified by their N-terminal peptides. Our data constitute the largest data set currently available for protein N-termini of prokaryotic photosynthetic organisms.