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.     

Alignment and statistical difference analysis of complex peptide data sets generated by multidimensional LC-MS

A method for high-resolution proteomics analyses of complex protein mixtures is presented using multidimensional HPLC coupled to MS (MDLC-MS). The method was applied to identify proteins that are differentially expressed during fruit ripening of tomato. Protein extracts from red and green tomato fruits were digested by trypsin. The resulting highly complex peptide mixtures were separated by strong cation exchange chromatography (SCX), and subsequently analyzed by RP nano-LC coupled to quadrupole-TOF MS. For detailed quantitative comparison, triplicate RP-LC-MS runs were performed for each SCX fraction. The resulting data sets were analyzed using MetAlign software for noise and data reduction, multiple alignment and statistical variance analysis. For each RP-LC-MS chromatogram, up to 7000 mass components were detected. Peak intensity data were compared by multivariate and statistical analysis. This revealed a clear separation between the green and red tomato samples, and a clear separation of the different SCX fractions. MS/MS spectra were collected using the data-dependent acquisition mode from a selected set of differentially detected peptide masses, enabling the identification of proteins that were differentially expressed during ripening of tomato fruits. Our approach is a highly sensitive method to analyze proteins in complex mixtures without the need of isotope labeling.     

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.     

Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome

Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.     

Identification and quantification of differentially expressed proteins in E-cadherin deficient SCC9 cells and SCC9 transfectants expressing E-cadherin by dimethyl isotope labeling, LC-MALDI MS and MS/MS

A strategy based on isotope labeling of peptides and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS) has been employed to accurately quantify and confidently identify differentially expressed proteins between an E-cadherin-deficient human carcinoma cell line (SCC9) and its transfectants expressing E-cadherin (SCC9-E). Proteins extracted from each cell line were tryptically digested and the resultant peptides were labeled individually with either d(0)- or d(2)-formaldehyde. The labeled peptides were combined and the peptide mixture was separated and fractionated by a strong cation exchange (SCX) column. Peptides from each SCX fraction were further separated by a microbore reversed-phase (RP) LC column. The effluents were then directly spotted onto a MALDI target using a heated droplet LC-MALDI interface. After mixing with a MALDI matrix, individual sample spots were analyzed by MALDI quadrupole time-of-flight MS, using an initial MS scan to quantify the dimethyl labeled peptide pairs. MS/MS analysis was then carried out on the peptide pairs having relative peak intensity changes of greater than 2-fold. The MS/MS spectra were subjected to database searching for protein identification. The search results were further confirmed by comparing the MS/MS spectra of the peptide pairs. Using this strategy, we detected and compared relative peak intensity changes of 5480 peptide pairs. Among them, 320 peptide pairs showed changes of greater than 2-fold. MS/MS analysis of these changing pairs led to the identification of 49 differentially expressed proteins between the parental SCC9 cells and SCC9-E transfectants. These proteins were determined to be involved in different pathways regulating cytoskeletal organization, cell adhesion, epithelial polarity, and cell proliferation. The changes in protein expression were consistent with increased cell-cell and cell-matrix adhesion and decreased proliferation in SCC9-E cells, in line with E-cadherin tumor suppressor activity. Finally, the accuracy of the MS quantification and subcellular localization for 6 differentially expressed proteins were validated by immunoblotting and immunofluorescence assays.     

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.     

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.     

Proteomic analysis of cold adaptation in a Siberian permafrost bacterium--Exiguobacterium sibiricum 255-15 by two-dimensional liquid separation coupled with mass spectrometry

Bacterial cold adaptation in Exiguobacterium sibiricum 255-15 was studied on a proteomic scale using a 2-D liquid phase separation coupled with MS technology. Whole-cell lysates of E. sibiricum 255-15 grown at 4 degrees C and 25 degrees C were first fractionated according to pI by chromatofocusing (CF), and further separated based on hydrophobicity by nonporous silica RP HPLC (NPS-RP-HPLC) which was on-line coupled with an ESI-TOF MS for intact protein M(r) measurement and quantitative interlysate comparison. Mass maps were created to visualize the differences in protein expression between different growth temperatures. The differentially expressed proteins were then identified by PMF using a MALDI-TOF MS and peptide sequencing by MS/MS with a MALDI quadrupole IT TOF mass spectrometer (MALDI-QIT-TOF MS). A total of over 500 proteins were detected in this study, of which 256 were identified. Among these proteins 39 were cold acclimation proteins (Caps) that were preferentially or uniquely expressed at 4 degrees C and three were homologous cold shock proteins (Csps). The homologous Csps were found to be similarly expressed at 4 degrees C and 25 degrees C, where these three homologous Csps represent about 10% of the total soluble proteins at both 4 degrees C and 25 degrees C.     

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.     

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 .     

Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis

A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.     

A solid phase extraction-based platform for rapid phosphoproteomic analysis

Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC–MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantitate peptides harboring >8000 unique phosphorylation sites in yeast in 12 h of instrument analysis time and examine the impact of enzyme choice and instrument platform.     

Phosphopeptides enrichment using on-line two-dimensional strong cation exchange followed by reversed-phase liquid chromatography/mass spectrometry

We have developed a method to isolate and enhance the detection of phosphopeptides using liquid chromatography (LC)/mass spectrometry on a tryptic-digested protein sample. The method uses an on-line two-dimensional chromatography approach that consists of strong cation exchange (SCX) followed by reversed-phase (RP) chromatography with mass spectrometric detection. At pH 2.6 or lower, tryptic phosphopeptides are not retained during the first-dimension SCX chromatography step. Thus the capture of these peptides in the flow-through by the second-dimension RP trap can dramatically reduce the complexity of the phosphopeptide chromatography, resulting in little or no suppression of the signal often caused by the coeluting nonphosphorylated peptides. The method provides higher phosphopeptide recovery and less nonspecific biding of acidic peptides than the commonly used enrichment methods, such as immobilized metal affinity chromatography. Since the widely adopted multidimensional LC strategy in shotgun proteomics uses a similar SCX-RP approach, the method can be adapted to detect and characterize phosphopeptides from a complex mixture in a single experiment. Limitations of the method are also discussed.     

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.     

Comparative analysis of OFFGel,strong cation exchange with pH gradient,and RP at high pH for first‐dimensional separation of peptides from a membrane‐enriched protein fraction

The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel‐based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX‐pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first‐dimensional separation of peptides using a detergent phase (Triton X‐114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX‐pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with “membrane” ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX‐pG and OFFGel techniques.     

Differential Proteome Analysis: Two-Dimensional Nano-LC/MS of E. Coli Proteome Grown on Different Carbon Sources

Different sugars provided to bacteria as single sources of carbon and energy require the induction of different metabolic enzymes, transporters, and uptake systems in order to support growth and cell survival. Using a nano–high-performance liquid chromatography/mass spectrometry (nano-HPLC/MS) system we constructed comprehensive peptide maps for Escherichia coli grown with either lactose or glucose in minimal medium. Digested bacterial samples were separated in a two-dimensional manner by combining strong cation exchange (SCX) and reversed-phased (RP) chromatography. Peptides were eluted online to an iontrap MS instrument and further analyzed by tandem MS fragmentation. Bacterial proteins originating from the differing samples were analyzed by searching the Swiss Prot Database. Data are presented that show the ability to detect several hundred different proteins significantly expressed under both conditions. Several enzymes and binding proteins related to the lactose metabolism were only identified in the sample grown with this carbon source.     

Analysis of human liver proteome using replicate shotgun strategy

In this study, a liquid-based shotgun strategy was used to comprehensively identify the expression of human liver proteome. Proteins were extracted from human liver tissue and digested in-solution. The tryptic digest mixture was desalted and separated by off-line strong cation exchange (SCX) chromatography with a 60-min elution. The MS/MS spectra were acquired in data-dependent mode after an RP chromatographic separation combined with linear IT MS analysis. To obtain the most comprehensive human liver proteome, each SCX fraction was run six times in RPLC MS/MS manner. Finally, more than 6,000,000 MS/MS spectra were collected. Using a relatively strict filter criteria, 24,311 proteins (48.42% of the predicted human proteome from human International Protein Index (IPI) protein database 3.07) corresponding to 13,150 nonredundant proteins were successfully identified, in which 7001 proteins (53.24%) were identified by two or more peptides, which could be considered as a high-confident dataset. Among the 6149 proteins (46.76%) identified by single peptide, 3812 proteins (61.99%) were detected more than twice in six repeated runs. Comparative analysis between different runs shows that the overlap of identified proteins between any two runs ranged from 25 to 44%. Of the nonredundant proteins identified, 8919 proteins (67.83%) were detected more than twice and 4231 proteins (32.17%) were detected only once in six RPLC MS/MS runs. The Gene Ontology annotation shows that the identified proteins come from various subcellular components. In addition, a large number of low abundant proteins were identified. The dynamic range of the approach reached at least nine orders of magnitude by estimating the concentration of proteins.     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.     

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.     

Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC-MS/MS

In this study we use replicate 2D-LC-MS/MS analyses of crude membranes from B cells derived from a patient with chronic lymphocytic leukemia (CLL) to examine the protein expression profile of CLL B cells. Protein identifications made by replicate 2D-LC-MS/MS analysis of tryptic peptides from detergent solubilized B cell membrane proteins, as well as replicate LC-MS/MS analysis of single off-line strong cation exchange chromatography (SCX) fractions, were analyzed. We show that despite the variance in SCX, capillary LC, and the data-dependent selection of precursor ions, an overlap of 64% between proteins identified in replicate runs was achieved for this system.