Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Protein- versus peptide fractionation in the first dimension of two-dimensional high-performance liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry for qualitative proteome analysis of tissue samples

The availability of robust and highly efficient separation methods represents a major requirement for proteome analysis. This study investigated the characteristics of two different gel-free proteomic approaches to the fractionation of proteolytic peptides and intact proteins, respectively, in a first separation dimension. Separation and mass spectrometric detection by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) were performed at the peptide level in both methods. Bottom-up analysis (BU) was carried out employing well established peptide fractionation in the first separation dimension by strong cation-exchange chromatography (SCX), followed by ion-pair reversed-phase chromatography (IP-RPC) in the second dimension. In the semi-top-down approach (STD), which involved intact protein fractionation in the first dimension, the separation mode in both dimensions was IP-RPC utilizing monolithic columns. Application of the two approaches to the proteome analysis of proteins extracted from a tumor tissue revealed that the BU method identified more proteins (1245 in BU versus 920 in STD) while STD analysis offered higher sequence coverage (14.8% in BU versus 17.5% in STD on average). The identification of more basic and larger proteins was slightly favored in the BU approach, most probably due to higher losses of these proteins during intact protein handling and separation in the STD method. A significant degree of complementarity was revealed by an approximately 33% overlap between one BU and STD replicate, while 33% each of the protein identifications were unique to both methods. In the STD method, peptides obtained upon digestion of the proteins contained in fractions of the first separation dimension covered a broad elution window in the second-dimension separation, which demonstrates a high degree of “pseudo-orthogonality” of protein and peptide separation by IP-RPC in both separation dimensions.     

Fractionation of phosphopeptides on strong anion‐exchange capillary trap column for large‐scale phosphoproteome analysis of microgram samples

It is one of the key issues to develop powerful fractionating method to increase the identification of the low‐abundance phosphopeptides. In this study, a semi‐online 2‐D LC separation strategy based on three‐step fractionation of the enriched peptides on strong anion‐exchange trap column was developed. It was demonstrated that the sensitivity and phosphoproteome coverage obtained by this fractionating method with strong anion‐exchange trap column is much higher than those by the conventional methods based on C18 trap column. In addition, when the same amount of sample was loaded, the number of identified phosphopeptides had increased 108%. Combination of this three‐step fractionation method with RPLC‐MS/MS analysis by 300 min RP‐gradient separation was applied to phosphoproteome analysis of human liver proteins, and 853 unique phosphopeptides was positively identified from 500 μg tryptic digest of human liver proteins. After three cycles' consecutive analyses, 1554 unique phosphopeptides and 1566 phosphorylated sites were totally identified from 735 phosphorylated proteins at a false discovery rate of <1% in about 54 h of analysis time.     

Evaluation of different two‐dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large‐scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide‐level fractionation, the first dimension of separation was performed with high‐pH reversed‐phase chromatography (pH‐RP) and strong cation exchange chromatography (SCX). Regarding protein‐level fractionation, C₈ protein reversed‐phase (C₈‐RP Prot) and high‐recovery protein reversed‐phase (hr‐RP Prot) were used instead. The second dimension consisted of a reversed‐phase nano‐HPLC on‐Chip coupled to an electrospray ionization quadrupole time‐of‐flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide‐level pH‐RP and the hr‐RP Prot protein‐level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide‐level SCX, with 509 proteins identified, was the worst method. Copyright © 2010 John Wiley & Sons, Ltd.     

Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO(2) pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.     

Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase,strong cation exchange,weak anion exchange,and fused‐core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C₁₈ at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C₁₈ at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.     

Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins. Analysis of tryptic peptides was accomplished with microfluidic HPLC-Chip/MS system. Results indicate that high-abundant protein depletion combined with RP fractionation of plasma showed an improved dynamic range for proteomic analysis and enabled the identification of low-abundant plasma proteins.     

Trace LC/MS quantitative analysis of polypeptide biomarkers: impact of 1-D and 2-D chromatography on matrix effects and sensitivity

We investigated the impact of one dimension (single reverse phase (RP) column) and two dimension (two different RP columns) chromatographic methods on SIM (MS) and multiple reaction monitoring (MRM; MS/MS) performance from human plasma. We find that MRM analysis is clearly preferable for 1-D applications; however, implementation of SIM detection in conjunction with 2-D separation technique resulted in an over 60-fold increase in analyte peak area and improved S/N compared to MRM for our analyte, human C-peptide. Implementation of a 2-D RP-RP technique with SIM detection is capable of eliminating matrix effects and greatly increases signal response and data quality. For two large peptides in complex biological samples, we found that a 2-D approach performed better than high quality sample preparation together with 1-D chromatography and MRM, even on a high-end mass spectrometer.     

On-Line multitasking analytical proteomics: how to separate, reduce, alkylate and digest whole proteins in an on-Line multidimensional chromatography system coupled to MS

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.     

Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success

In high-throughput proteomics, the bottom-up approach has become a widely used method for the identification of proteins that is based on tryptic peptide MS/MS analysis. Separation methodologies that use IEF of tryptic peptides have recently been introduced and provide an extra dimension of peptide separation. In addition to its great fractionation capability, tryptic peptide prefractionation by IEF can also increase the protein identification success. The pI information of the peptide gained can be successfully used in a post-database search filtering step. We introduce a filtering algorithm that is based on the comparison of the experimental and theoretical pI's to validate peptide identifications by MS/MS data search engines.     

Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.     

Ion-pair Reversed-phase×Low-pH Reversed-phase Two-dimensional Liquid Chromatography for In-depth Proteomic Profiling

High-resolution liquid chromatography separation is essential to in-depth proteomic profiling of complex biological samples. Herein, we established an ion-pair reversed-phase×reversed-phase two-dimensional liquid chromatography (IPRP×RP 2DLC) strategy for comprehensive proteomic analysis. Both RPLC separation dimensions were performed at low pH, with trifluoroacetic acid(TFA) and formic acid(FA) as mobile phase addictive, respectively. As the good separation resolution offered by ion-pairing effect of TFA, the fractionation efficiency was greatly improved with 74.0% peptides identified in just one fraction. Comparing with conventional high pH RP fractionation, the overall separation rate of IPRP was about 1.6 times that of high-pH RP, which increased the number of identified peptides by 21%. Further, 2169 proteins and 8540 peptides were confidently identified from crude serum sample by our IPRP×RP 2DLC strategy, exhibiting great potential in clinical proteomics in the future.     

Micro-proteome analysis using micro-chromatofocusing in intact protein separations

Multi-dimensional liquid-based separation is required for fractionation and mapping of complex protein mixtures from cells. A method that has been used as the first dimension in such separations is chromatofocusing (CF), which is based on generating a pH gradient on an anion exchange column. The use of pH in the first dimension is essential where pH is a fundamental property of proteins and can provide information on post-translationally modified forms of a protein. In this work, a micro-chromatofocusing technique is introduced which can separate microgram levels of proteins from cell lysates for further analysis by LC-MS/MS. It is shown that this method can analyze 10 microg of sample and detect nearly 700-800 proteins from ovarian cancer cell line lysates.     

A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The “GELFrEE” (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5–25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.     

Evaluation of peptide fractionation strategies used in proteome analysis

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2‐D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong‐cation exchange, hydrophilic interaction chromatography, alkaline‐RP and solution isoelectric focusing, which can be used prior to LC‐MS/MS analysis, were compared. Strong‐cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide‐based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.