Large scale depletion of the high-abundance proteins and analysis of middle- and low-abundance proteins in human liver proteome by multidimensional liquid chromatography

An unbiased method for large-scale depletion of high-abundance proteins and identification of middle- or low-abundance proteins by multidimensional LC (MDLC) was demonstrated in this paper. At the protein level, the MDLC system, coupling the first dimensional strong cation exchange (SCX) chromatography with the second dimensional RP-HPLC, instead of immunoaffinity technology, was used to deplete high-abundance proteins. Sixty-two fractions from SCX were separated further by RPLC. UV absorption spectra were observed to differentiate high-abundance proteins from middle- or low-abundance proteins. After the depletion of high-abundance proteins, middle- or low-abundance proteins were enriched, digested, and separated by online 2D-micro-SCX/cRPLC. The eluted peptides were deposited on the MALDI target and detected by MALDI-TOF/TOF MS. This depletion strategy was applied to the proteome of the normal human liver (NHL) provided by the China Human Liver Proteome Project (CHLPP). In total, 58 high-abundance proteins were depleted in one experiment. The strategy increases greatly the number of identified proteins and around 1213 proteins were identified, which was about 2.7 times as that of the nondepletion method.     

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.     

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.     

Recent development of multi-dimensional chromatography strategies in proteome research

As a complementary approach to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), multi-dimensional chromatography separation methods have been widely applied in all kinds of biological sample investigations. Multi-dimensional liquid chromatography (MDLC) coupled with bio-mass spectrometry (MS) is playing important roles in proteome research due to its high speed, high resolution and high sensitivity. Proteome analysis strategies mainly include bottom-up and top-down approaches which carry out biological sample separation based on peptide and protein levels, respectively. Electrophoretic methods combined with liquid chromatography like IEF-HPLC and HPLC-SDS-PAGE have been successful applied for protein separations. As for MDLC strategy, ion-exchange chromatography (IEX) together with reversed phase liquid chromatography (RPLC) is still a most widely used chromatography in proteome analysis, other chromatographic methods are also frequently used in protein pre-fractionations, while affinity chromatography is usually adopted for specific functional protein analysis. Recent MDLC technologies and applications to variety of proteome analysis have been achieved great development. A digest peptide-based approach as so-called "bottom-up" and intact protein-based approach "top-down" analysis of proteome samples were briefly reviewed in this paper. The diversity of combinations of different chromatography modes to set up MDLC systems was demonstrated and discussed. Novel developments of MDLC techniques such as high-abundance protein depletion and chromatography array were also included in this review.     

Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry

The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.     

Development of a highly efficient 2-D system with a serially coupled long column and its application in identification of rat brain integral membrane proteins with ionic liquids-assisted solubilization and digestion

Two dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-HPLC-ESI-MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was ～55 protein groups/h, nearly 2-fold of that for SC-2D (～28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtained by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.     

Developing a strong anion exchange/RP (SAX/RP) 2D LC system for high‐abundance proteins depletion in human plasma

Human plasma is dominated by high‐abundance proteins which severely impede the detection of low‐abundance proteins. Unfortunately, now there is no efficient method for large‐scale depletion of high‐abundance proteins in human plasma. In this study, we developed a new strategy, strong anion exchange (SAX)/RP 2D LC system, which has potential for large‐scale depletion of high‐abundance proteins in human plasma. Separation gradients of the system were optimized to ensure an extensive separation of plasma proteins. Plasma was fractionated into 67 fractions by SAX. All these fractions were subjected a thorough separation by the 2D RPLC and 66 peaks with high UV absorption (>20 mAU) at 215 nm were collected. Proteins in these peaks were identified by LC‐MS/MS analysis. Results showed that 83 proteins could be identified in these peaks, 68 among them were reported to be high‐ or middle‐abundance proteins in plasma. All these proteins had definite retention times and were mapped in the 2D SAX‐RP system, which resulted in accurate depletion of high‐abundance proteins with ease. Our studies provide a convenient and effective method for large‐scale depletion of high‐abundance proteins and in‐depth research in human plasma proteomics.     

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 .     

Fully automated multifunctional ultrahigh pressure liquid chromatography system for advanced proteome analyses

A multifunctional liquid chromatography system that performs 1-dimensional, 2-dimensional (strong cation exchange/reverse phase liquid chromatography or SCX/RPLC) separations and online phosphopeptide enrichment using a single binary nanoflow pump has been developed. With a simple operation of a function selection valve equipped with a SCX column and a TiO(2) (titanium dioxide) column, a fully automated selection of three different experiment modes was achieved. Because the current system uses essentially the same solvent flow paths, the same trap column, and the same separation column for reverse-phase separation of 1D, 2D, and online phosphopeptides enrichment experiments, the elution time information obtained from these experiments is in excellent agreement, which facilitates correlating peptide information from different experiments. The final reverse-phase separation of the three experiments is completely decoupled from all of the function selection processes; thereby salts or acids from SCX or TiO(2) column do not affect the efficiency of the reverse-phase separation.     

Depletion of high-abundance proteins from serum by immunoaffinity chromatography: a MALDI-FT-MS study

Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-FT-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks.     

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.     

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.     

Enhanced separation and characterization of deamidated peptides with RP-ERLIC-based multidimensional chromatography coupled with tandem mass spectrometry

Deamidation of asparaginyl residues in proteins produces a mixture of asparaginyl, n-aspartyl, and isoaspartyl residues, which affects the proteins' structure, function, and stability. Thus, it is important to identify and quantify the products to evaluate the effects in biological systems. It is still a challenging task to distinguish between the n-Asp and isoAsp deamidation products in a proteome-wide analysis because of their similar physicochemical properties. The quantification of the isomeric deamidated peptides is also rather difficult because of their coelution/poor separation in reverse-phase liquid chromatography (RPLC). We here propose a RP-ERLIC-MS/MS approach for separating and quantifying on a proteome-wide scale the three products related to deamidation of the same peptide. The key to the method is the use of RPLC in the first dimensional separation and ERLIC (electrostatic repulsion-hydrophilic interaction chromatography) in the second, with direct online coupling to tandem MS. The coelution of the three deamidation-related peptides in RPLC is then an asset, as they are collected in the same fraction. They are then separated and identified in the second dimension with ERLIC, which separates peptides on the basis of both pI and GRAVY values. The coelution of the three products in RPLC and their efficient separation in ERLIC were validated using synthetic peptides, and the performance of ERLIC-MS/MS was tested using peptide mixtures from two proteins. Applying this sequence to rat liver tissue, we identified 302 unique N-deamidated peptides, of which 20 were identified via all three deamidation-related products and 70 of which were identified via two of them.     

Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy

In proteomics, a digested cell lysate is often too complex for direct comprehensive mass spectrometric analysis. To reduce complexity, several peptide separation techniques have been introduced including very successful two-dimensional liquid chromatography (2D-LC) approaches. Here, we assess the potential of zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) as a first dimension for the analysis of complex peptide mixtures. We show that ZIC-HILIC separation is dramatically dependent on buffer pH in the range from 3 to 8, due to deprotonation of acidic amino acids. ZIC-HILIC exhibits a mixed-mode effect consisting of electrostatic and polar interactions. We developed a 2D-LC system that hyphenates ZIC-HILIC off-line with reversed-phase (RP). The two dimensions are fairly orthogonal, and the system performs very well in the analysis of minute amounts of complex peptide mixtures. Applying this method to the analysis of 10 mug of a cellular nuclear lysate, we were able to confidently identify over 1000 proteins. Compared to strong cation exchange chromatography (SCX), ZIC-HILIC shows better chromatographic resolution and absence of clustering of prevalent +2 and +3 charged peptides. At pH 3, ZIC-HILIC separation allows best orthogonality with RP and resembles conventional SCX separation. A significant enrichment of N-acetylated peptides in the first fractions is observed at these conditions. ZIC-HILIC separation at high pH (6.8 and 8), however, enables better chromatography, resulting in more comprehensive data acquisition. With this extended flexibility, we conclude that ZIC-HILIC is a very good alternative for the more conventional SCX in multidimensional peptide separation strategies.     

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.     

Assessment approach for evaluating high abundance protein depletion methods for cerebrospinal fluid (CSF) proteomic analysis

Optimal proteomic analysis of human cerebrospinal fluid (CSF) requires depletion of high-abundance proteins to facilitate observation of low-abundance proteins. The performance of two immunodepletion (MARS, Agilent Technologies and ProteoSeek, Pierce Biotechnology) and one ultrafiltration (50 kDa molecular weight cutoff filter, Millipore Corporation) methods for depletion of abundant CSF proteins were compared using a graphical method to access the depth of analysis using "marker proteins" with known normal concentration ranges. Two-dimensional LC/MS/MS analysis of each depleted sample yielded 171 and 163 unique protein identifications using the MARS and ProteoSeek immunodepletion methods, respectively, while only 46 unique proteins were identified using a 50 kDa molecular weight cutoff filter. The relative abundance of the identified proteins was estimated using total spectrum counting and compared to the concentrations of 45 known proteins in CSF as markers of the analysis depth. Results of this work suggest a clear need for methodology designed specifically for depletion of high-abundance proteins in CSF, as depletion methods designed to deplete high-abundance serum proteins showed little improvement in analysis depth compared to analysis without depletion. The marker protein method should be generally useful for assessing depth of analysis in the comparison of proteomic analysis methods.     

High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis

Orthogonal high-resolution separations are critical for attaining improved analytical dynamic range and protein coverage in proteomic measurements. High-pH reversed-phase liquid chromatography (RPLC), followed by fraction concatenation, affords better peptide analysis than conventional strong cation-exchange chromatography applied for 2D proteomic analysis. For example, concatenated high-pH RPLC increased identification of peptides (by 1.8-fold) and proteins (by 1.6-fold) in shotgun proteomics analyses of a digested human protein sample. Additional advantages of high-pH RPLC with fraction concatenation include improved protein sequence coverage, simplified sample processing and reduced sample losses, making this an attractive alternative to strong cation-exchange chromatography in conjunction with second-dimension low-pH RPLC for 2D proteomics analyses.     

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS

Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm×150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC-MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.     

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.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.