Briefings in Functional Genomics and Proteomics Advance Access originally published online on November 22, 2006
Briefings in Functional Genomics and Proteomics 2006 5(4):249-260; doi:10.1093/bfgp/ell034
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HPLC techniques for proteomics analysis—a short overview of latest developments
Corresponding author. Goran Mitulovi
, Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Wien, Austria. E-mail: mitulovic{at}imp.univie.ac.at |
ABSTRACT |
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TOP
ABSTRACT
INTRODUCTIONDue to the complex nature of the proteome, instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation and detection becomes urgent for the identification of peptides and proteins. Newly developed techniques and equipment for separation and detection, such as nano-HPLC and multidimensional HPLC for protein and peptide separation, enabled proteomics to experience dynamic growth during the past few years. In any proteomic analysis the most important and sometimes most difficult task is the separation of the complex mixture of proteins or peptides. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, which would need far more space than available here.
Keywords: proteomics, liquid chromatography-mass spectrometry, multidimensional separation, post-translational modifications
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INTRODUCTION |
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The term ‘proteome’ appeared in the literature during the mid 1990s [1] describing the ensemble of proteins in a living organism or a living cell, related to the genome expressing these proteins. Proteomics, defined as the study of proteomes, started its growth in the mid 1990s and has seen a tremendous development up to the present day.
In the early days of proteomics, separation of proteins and peptides was performed by two-dimensional gel-electrophoresis (2DGE), two-dimensional electrophoresis (2DE) and detection mainly with matrix assisted laser desorption ionization mass spectrometry (MALDI MS) [2, 3]. In the beginning, it was simply analyzing proteins, and over the years it has expanded into profiling, structural and functional proteomics including a wide range of technologies used for analysis.
Briefly, profiling proteomics attempts to profile the proteins differently expressed between two different samples. This approach is used to show the difference in expressing proteins in cells or an organism being in two different states, e.g. a healthy state and a diseased state. Functional proteomics is applied when searching for protein functions on post-translational modified proteins, or to study the interaction of proteins with substrates and small molecules.
Post-translational modifications (PTMs) are the key to understanding the functions and roles of proteins in a living organism. Since the proteins need to interact with other molecules to perform their role in an organism, knowing the reaction pathways in which they are involved helps to understand the ‘wiring diagram’ of a cell. Also, profiling proteomics can depict the changes in a proteome during an illness or a stress and so helps in understanding of processes such as signalling during diseases like cancer.
Structural proteomics focuses at the tertiary structure of proteins and protein complexes with small molecules and other proteins.
Protein identification by MS has become routine and the method of choice for proteomics studies. The two main approaches are MALDI-time-of-flight (TOF)-MS and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedures to obtain peptide fingerprinting profiles. Details of these methods have been reviewed in many articles and publications [4, 5].
Sample separation in the case of MALDI-TOF-MS is off-line, and the peptides are spotted on a target for subsequent MS analysis. ESI-MS, however, is an online HPLC-MS method, and the challenge is to increase the sensitivity of detection by using separation columns of smaller internal diameters (IDs) and lower flow rates. This is no simple task, since the smaller column IDs and lower flow rates also result in increased delay times, void volumes become critical and can also nullify the separation efforts, and the additional problem of carry-over may occur [6, 7].
The majority of the proteomics literature describes efforts for improving resolution of the separation and increasing the sequence coverage of a protein by separating complex mixtures of tryptically digested proteins. Methods developed such as peak parking and multidimensional separations [8–13] have increased the sensitivity for sample analysis and enabled further insight into the protein sample.
However, sensitivity is the driving factor to develop HPLC columns of smaller and smaller diameter and to develop new types of stationary phases, which are capable of selectively binding certain types of analyte. Theoretically, reducing the ID of a column from d1 to d2 results in an enhanced sensitivity (f), given by:
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Belov et al. [14] were able to detect proteins at the zeptomole (10–21 moles) level using a 15 µm ID column of 80 cm length.
Here, the flow rates needed for operating such small ID columns decrease exponentially with column diameter. If, for example, a 1 mm ID HPLC column needed a flow rate of 50 µl/min, a column of 75 µm ID would need approximately 300 nl/min.
Lower flow rates require new instrumental design and new approaches to flow delivery. This article will discuss the latest HPLC techniques used for proteomics research.
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INSTRUMENTATION |
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Instrumentation for HPLC research in proteomics does not differ from conventional HPLC instrumentation. Pumping systems, separation columns and detectors used for proteomics research are also used for conventional analysis. The difference, however, is the magnitude of the flow rate and therefore of the columns.
According to Chervet et al. [15], all system components should be downscaled, including flow rate, connecting tubing, detection and injection volumes. Samples for proteomic analysis are available in high amounts; however, the analytes are present in minute concentrations. Therefore, pumping systems were developed for providing flow rates in the nl/min range. There are commercially available HPLC systems both with and without flow splitting. It would take too much space to discuss the systems available on the market, and the reader is advised to check the home pages of the companies listed in the appendix for detailed information on each system. Briefly, in systems using flow splitting, a high pump flow rate of approx. 200–300 µl/min is split into the column flow rate of approx. 100–300 nl/min and the rest is directed into the waste. Chromatographic systems without flow splitting use syringe pumps to deliver the mobile phase to the column. While the majority of systems in use employ split flow, the systems without flow splitting are surprisingly under-represented. Both approaches, split flow and non-split flow, have pros and cons and both can be successfully applied for sample analysis.
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SEPARATION COLUMNS |
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Separation columns for proteomics research usually use the same stationary phase as for conventional HPLC. However, the ever-increasing demand for more selectivity, sensitivity and specificity has led to three main developments: first, improvements in existing reversed-phase (RP) stationary phases, allowing operation with very low or no trifluoroacetic acid (TFA), as for PepMap® columns; secondly, the emergence of monolithic columns, whose separation medium consists of a continuous, rigid polymeric rod with a porous structure, enabling faster separations [16–23]; and thirdly, the development of miniaturized columns in a chip format.
The separation columns as well as trap columns or precolumn are of great interest. Trap columns are usually used in column-switching mode and enable injections of high volumes of highly diluted samples. Several groups reported the use of trap columns [16, 24, 25] for analysis of large proteomics samples. Relatively short trap columns have larger IDs compared to the separation column (1–5 mm length and 300 µm ID versus 75 µm) and enable fast sample loading and desalting. However, the trap columns must show high load capacity and low void volumes. Schaefer et al. [7] and Mitulovic et al. [6] investigated the influence of column loading and void volumes on the quality of separation by nano-HPLC.
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DETECTORS FOR HPLC |
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All detector types used for conventional HPLC are applicable for proteomics analysis, but one cannot differentiate by UV spectra alone whether two or more peptides are co-eluting. While the UV detector is mainly used for quality-controlling of the separation (void volume, impurities, base line and gradient stability) and for tracing fractions when the sample is being fractionated, the mass spectrometer is the workhorse detector for proteomics. MALDI and electrospray detectors are used for both characterization and quantitation of separated analytes. The newly developed Orbitrap® mass spectrometer and TOF-TOF mass spectrometers along with Fourier Transform (FT) mass spectrometers have contributed greatly to generating more accurate and more sensitive results. Additionally, new and fast separation media like those in monolithic columns and ultra-performance chromatography need detectors that can respond quickly due to reduced peak width during very fast separations [16–23, 26, 27].
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HPLC APPLICATIONS FOR PROTEOMICS |
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Each type of HPLC has undergone significant developments lately, but reversed-phase chromatography (RPC) is still the most widely used chromatography for sample preparation or sample separation. RPC, based on the interaction between the sample with a hydrophobic stationary phase and a polar hydrophilic mobile phase, is the most used separation technique for proteomics, and was introduced in 1976 for peptide separation [28]. For proteomics research, separations of tryptically digested peptides on nano columns with ID
100 µm are performed. By combining RP separation column with columns such as ion exchange (IEX) or immobilized metal-ion affinity chromatography (IMAC), the amount and quality of analytical information is highly increased. Peptides which have been enriched, trapped, pre-fractionated or desalted on other column types, are eluted and separated on a RP column prior to detection by MS. |
ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS |
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The proteome was termed as protein complement expressed by a genome [1, 26, 27]. A definition like this implies a static nature, which in reality is not the case. The proteome is highly dynamic, its state depending on physiological conditions: the abundance and type of expressed proteins is not always the same, and they change with the physiological state of the cell or the tissue where they are expressed. Currently, more than 100 different post-translational modifications (PTMs) have been described [29] and it is very likely that this number will increase. Analysis of PTMs is, however, more difficult than for non-modified proteins. This is for two main reasons: (i) proteins are modified to a low stoichiometry; and (ii) the peptide-modification bond is often very labile. A novel approach for the analysis of PTMs using a monolithic column has been described by Hosoya et al. [30]. This separation system employs water as mobile phase and does not need any organic solvents, thus reducing the risk of removing or masking existing PTMs on a peptide for detection. The new stationary phase used for analysis is based on surface-modified polymer-based media. The authors describe the formation or disintegration of the complex between two polymeric selectors (polyacrylamide and poly-(methacrylic acid)) due to changes in temperature or the pH value of the mobile phase. The matrix becomes hydrophobic when the complex between the polymers is formed and hydrophilic when it disintegrates.
Despite the fact that several hundred PTMs are known, only a few of them have been shown to be reversible and therefore of potentially regulatory importance in biological systems and processes. Of these reversible PTMs, protein phosphorylation is the most studied and is the best understood regarding both enzymes involved in phosphorylation and dephosphorylation reactions. The most common type of phosphorylation is the formation of phosphate ester bonds with hydroxyl groups in the side chains of serine, threonine and tyrosine residues. Two types of enzyme, protein kinases and protein phosphatases, catalyse the processes of phosphorylation and dephosphorylation respectively, and their structures and the reaction mechanism are well studied.
The review of HPLC applications for the analysis of the numerous PTMs would require far more space than is available here, and that is why the reviewer will focus on HPLC analysis of phosphorylated proteins and peptides. Sample preparation and HPLC separation of this group of proteins and peptides can involve almost all HPLC technologies presently known: RPC, affinity chromatography, IEX chromatography, size exclusion chromatography, the use of monolithic stationary phases and miniaturized separation systems, etc.
Phosphorylation occurs at low stoichiometry and the analysis of phosphorylated proteins is quite challenging for both chromatography and MS. Proteins of high abundance generate an overwhelming amount of peptides after proteolytic digestion, and these tend to mask low-abundant peptides in the sample. Also, low-abundant proteins or peptides often co-elute with high-abundant peptides and cannot be identified on the UV trace. The first step is always the reduction of sample complexity. Frequently, proteins are not only phosphorylated at low stoichiometry but also at multiple sites (this gives a rise to differentially phosphorylated forms of the same protein); it is difficult to isolate quantities of such a protein to be analysed by even the most sensitive LC-MS/MS method.
Commonly used techniques for the separation of phosphorylated peptides are 2DE mapping on cellulose plates, one and two-dimensional high-resolution gel electrophoresis, RPC, IMAC and the combination of anion exchange chromatography and RPC. Connecting the HPLC system to the mass spectrometer enables analysis of highly complex samples without a need to perform radioactive labelling of the peptides.
RPC of phosphorylated peptides is simple, robust and reproducible. Unfortunately, the peak capacity of conventional RP separation is not as high as the peak capacity of 2D gels, and another problem is the use of stainless steel injectors, tubing, pump filters and column frits. Phosphopeptides show a high affinity for metal surfaces, often and readily forming complexes with the metal surfaces of HPLC apparatus. This kind of risk can be lowered if HPLC systems with polyether ether ketone (PEEK) or titanium injectors, filters and column frits are used. Despite these limitation factors, RPC is readily and widely used for separation of samples containing phosphopeptides because of its easy coupling with ESI-MS or with off-line fraction collection and MALDI-MS.
A very important factor that sometimes limits the use of RPC for phosphopeptide analysis, is the fact that very hydrophilic peptides cannot be trapped on RP separation columns and therefore will be lost during analysis. Very hydrophobic peptides will bind too well to the stationary phase, and will not elute until the higher concentrations of organic solvent in the mobile phase reach the column. A high percentage of organic compounds in the mobile phase will also elute a considerable amount of polymers and other impurities from the column, thus masking the signal from the peptides. Mitulovic et al. presented a poster at 53rd ASMS 2005 (publication in preparation) describing a method for improved trapping of hydrophilic phosphopeptides. Usually, TFA is used as an ion-pairing agent in the mobile phase during loading in order to better trap peptides on the pre-concentration column. However, the ionic strength is not enough for binding small and hydrophilic peptides. By using a mixture of three organic acids—formic acid (FA), TFA and heptafluoro butyric acid (HFBA)—it is possible to trap the peptides, induce longer retention times on the RP separation column and detect and analyse them by MS/MS. The observation is that not only hydrophilic peptides stick better on the column but also the hydrophobic peptides could be separated from the major impurities eluting with the late gradient [31].
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MULTIDIMENSIONAL SEPARATION TECHNIQUES |
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One-dimensional HPLC has been proved to be reproducible and effective for peptide and protein separation. However, its use in proteomics is restricted due to the sample complexity; after proteolytic digestion, the number of peptides needed to be separated reaches hundreds or thousands and this exceeds the peak capacity of most 1D-HPLC columns [32].
To improve resolution, multidimensional separation techniques have been introduced and the use of this approach has improved rapidly.
Theoretically, the peak capacity of the multidimensional separation system is given by the equation:
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In multidimensional separation approach, IEX chromatography is usually the first step preceding the nano RP–HPLC. But in specific applications, such as analysis of glycopeptides or phosphopeptides, other techniques are used, such as titanium columns or the IMAC enrichment of phosphopeptides.
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TITANIUM COLUMNS FOR SELECTIVE ENRICHMENT OF PHOSPHOPEPTIDES |
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The affinity of titanium oxide (TiO2) for organic phosphates was discovered more than 15 years ago but only recently this stationary phase was introduced for the purpose of selective enrichment of phosphopeptides [33–37]. Larsen et al. [38] reported improved trapping of phosphorylated peptides onto the self-packed titanium trap columns. This work describes the use of well-known MALDI matrix DHB (2,5-dihydroxybenzoic acid) as additive to the loading and washing phase for titanium column. The number of co-trapped acidic peptides was significantly reduced without influencing the separation efficiency of the RP separation column.
Elution of organic phosphates from the titanium column was also achieved by using borate buffers of high pH, which made the use of this column type unsuitable if combined with silica-based separation columns, due to the high pH of the eluting buffer. Additionally, borate buffers suffered from instability so that the pH value and the buffer composition were not constant. Kuroda et al. [39] developed a separation procedure where the titanium column is used online with a RP monolithic silica separation column. Here, automated column switching with MS detection of phosphopeptides was introduced and described. Phosphopeptides (in this case, tryptic peptides generated from ß-casein) were loaded onto a titanium trap column using an aqueous 0.1% TFA solution and eluted with 100 mM or 200 mM KH2PO4–K2HPO4 solution onto a monolithic silica trap column. Phosphate buffer for elution of phosphopeptides was used at pH 7.0 and did not deteriorate the silica-based separation column. In order to enable proper MS operation and detection of trapped phosphopeptides, phosphate buffer was washed out of the trap column by pumping aqueous 0.1% TFA through it. Free of phosphate buffer, the trap column was switched online with the separation column and phosphopeptides were separated, and detected by UV and MS.
Pinkse et al. [36, 37] reported some promising results for this type of stationary phase at the 52nd ASMS conference, and since then the number of publications where titanium was used increases rapidly. Pinkse describes the use of 100 µm ID columns packed with titanium, together with 50 µm C18 packed separation columns for the selective enrichment and separation of tryptic peptides from cGMP-dependent protein kinase (PKG). This enzyme is 153 kDa homodimeric protein acting directly downstream in the nitric oxide/cGMP-mediated signalling pathway, controlling a variety of cellular responses, including smooth muscle relaxation [40]. Upon activation, this protein undergoes autophosphorylation, and this process affects the kinetic properties of PKG. The authors showed that it is possible to retain and recover phosphopeptides at the low femtomol level and also retain and detect multiple phosphorylated peptides. Using described chromatographic methods, it was shown that previously uncharacterized phosphorylation on Ser-26 is actually not an autophosphorylation site in vitro, but is most likely due to the action of some other protein. Several new phosphorylation sites were also uncovered and documented.
Schlosser et al. [41] investigated the phosphorylation sites of the murine circadian protein period 2 (mPER2), one of the key components constituting the mammalian circadian clock, an endogenous timing system that adapts physiological processes to the varying demands of a day. They used specific enrichment of phosphopeptides on a titanium column and subsequently separation on a 25 µm ID C18 separation column. The setup of the nano-LC system has been described by Meiring et al. [42]. The small column ID allowed for flow rates as low as 25 nl/min, which strongly increased the sensitivity of the MS analysis. The phosphoprotein was digested with four different proteases: trypsin, elastase, proteinase K and thermolysin. Phosphopeptides were successfully trapped on a titanium column using highly acidic 30% acetonitrile, and the non-phosphorylated peptides (both acidic and basic) were not trapped. The author showed that the use of multiple digestion enzymes and a titanium column for specific isolation of phosphorylated peptides could significantly increase the number of phosphorylation sites detected.
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IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY FOR HPLC OF PHOSPHORYLATED PEPTIDES |
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Phosphorylated proteins and peptides show a high affinity towards metal ions and form quite stable complexes with these. By using this property of phosphorylated peptides, it is possible to selectively bind and enrich them on separation columns which have immobilized metal ions on their surface. The most frequently ions used are Fe3+, Ga2+, Ni2+, Cr2+, Al3+, Zn2+ and Cu2+. Metal cations are chelated by a multidentate ligand, which is immobilized onto a support material. Proteins are proteolytically digested and loaded onto the immobilized metal affinity column [43–45].
Unfortunately, it is not only the phosphopeptides which bind on to such a column, but also the acidic non-phospho peptides. In order to minimize non-specific binding of acidic peptides, Ficcaro et al. [46] converted all free carboxyl groups of the peptides in solution to the corresponding methyl esters and then analysed with IMAC and nano-HPLC-MS. Phosphopeptides were eluted from the IMAC column onto the C18 trap column using 50 mM Na2HPO4. The phosphate was washed out of the column using aqueous 0.1% formic acid, and then the trap column was switched in line with the separation column and MS. A total of 216 peptides, defining 383 phosphorylation sites, were identified. The coupling of the IMAC and RP trap and separation columns was automated and online, which enabled higher sample throughput. Additionally, this approach enables not only identification but also quantification of phosphopeptides if the methanol used for esterification is replaced by deuterated methanol.
Ren et al. presented separation of histidine-rich peptides from Saccharomyces cerevisae used in ethanol production with additional quantification by using a Cu2+-loaded IMAC column [45]. Another application for phosphorylation determination with IMAC columns was published by Catlin et al. [47], describing the selective enrichment of phosphopeptides from the tumour necrosis factor (TNF) signal transduction pathway using HeLa cells metabolically labelled with 15N-labelled amino acids. The authors chose this pathway since it is known that phosphorylation plays a key role in the transduction of the TNF signal through multiple protein complexes, and because the pathway is of great interest to many researchers due to its integral role in inflammation, apoptosis and cancer. Phosphopeptides are enriched using a Ga2+ IMAC column and subsequently separated on a C18 separation column. Authors found 223 phosphopeptides, of which 33 were up-regulated by 2-fold or more and 15 were down-regulated. In previous studies, 2-fold changes were found to be of great biological significance.
Wang et al. [48] described the use of automated IMAC analysis with a slightly modified esterification procedure for phosphopeptides. Here, the use of an automated approach increased the sample throughput and enabled faster screening of samples of interest.
Metal cations can be immobilized on different support materials, but mostly on silica or poly(styrene–divinylbenzene) particles. However, other media such as cellulose are also sometimes used. Feuerstein et al. [49] immobilized Fe3+ cations on cellulose and performed selective enrichment and separation of phosphorylated peptides. Rat ERK2 (extracellular signal regulated kinase 2) protein was tagged at its N-terminus with the GST epitope and phosphorylated in vitro. The authors were able to isolate the phosphorylated peptides after tryptic digestion of the protein.
A novel method for capturing and analysing phosphorylated peptides is described by Blacken et al. [32]. With this approach, phosphopeptides are captured as ternary complexes with Ga3+ or Fe3+ and N
,N-bis(carboxymethyl)lysine in solution and electrosprayed as doubly or triply charged ions into the MS.
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ION EXCHANGE CHROMATOGRAPHY—RP HPLC |
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Although IEX chromatography is routine for separation and purification of crude protein samples, it has been used only sporadically for peptide separations in combination with nano-HPLC for complex proteomic samples. In 2001, Washburn et al. [50] introduced the multidimensional separation for complex peptide samples by using a strong cation exchange (SCX) column for the separation of peptides in the first dimension. This approach, termed as multidimensional protein identification technology (MudPIT), involves a single biphasic column packed with SCX stationary phase, and C18 RP. It was possible to identify
25% of the complete yeast proteome (1484 proteins out of 6000 predicted ORFs) within a single experiment. However, this kind of separation suffers from a significant problem affecting MS—salt used for peptide elution from the SCX column cannot be washed into the waste, but elutes into the mass spectrometer, thus suppressing the ionization of the analytes. Mitulovi et al. [51] reported multidimensional separation of a tryptic digest of nuclear matrix protein preparation of Jurkat cells by using multistep salt injections for elution of peptide fractions from the SCX column onto the RP trap column. This approach enabled salt wash from the trap column, thus eliminating signal suppression and interaction in the mass spectrometer. Furthermore, the use of a ‘column-switching’ approach enabled the use of columns with different IDs and different chemistries. The authors were able to identify a total of 174 proteins, of which 46 have been identified with both 2D gels and 2D HPLC. Here, Na2PO4 buffer was used instead of the previously used ammonium acetate or ammonium formate buffers. However, the use of loop injections of potassium chloride or ammonium acetate resulted in band broadening and overlapping fractions, which complicated the identification and lead to multiple identifications of the same sample in several fractions. Improved peak shapes were observed when a continuous gradient was applied to the SCX column [52]. In contrast to the online methods, the ‘off-line’ methods separate the peptides by fraction collection and subsequent desalting and HPLC-MS/MS analysis. Wagner et al. [53] reported the ‘off-line’ 2D HPLC with a linear salt gradient applied instead of a stepwise salt injection approach, which increased the amount of recovered peptides by a factor of five. The authors identified a total of 95 proteins from digested yeast ribosomes, a figure higher than the number of available proteins in ribosomes. This can be explained by the fact that other proteins are associated with the ribosomes.
A significant number of phosphorylation sites were identified by using the ‘off–line’ continuous–gradient–SCX approach. Ballif et al. [54] reported over 500 phosphorylation sites in a tryptic digest of mouse brain tissue.
Winnik [55] described ‘online’ 2D separation of complex peptide mixtures by using two RP trap columns for higher sample throughput. Here, peptides were not eluted by a salt gradient or salt injections but by pH gradient, starting with sample loading onto the SCX column at pH 3.7, followed by raising the pH to 6.0, which resulted in decreased peptide binding capacity, thus facilitating peptide elution. This approach eliminated the use of NaCl or KCl for peptide elution and eliminated salt cluster in MS.
Multidimensional chromatography does not only imply the separation of peptides in the first dimension, the so-called ‘bottom-up’ approach, but also the ‘top-down’ approach, which is the separation of proteins in the first dimension [56–59] with subsequent RP separation and mass spectrometric detection of intact proteins. Millea et al. [56] demonstrated the use of multidimensional separation, combining anion exchange (AEX) with SCX and RP nano-HPLC for the comprehensive characterization of components within a complex protein mixture. The authors were able to identify co-translational modifications, PTMs and protein-processing events, and discriminate between protein isoforms.
Kislinger et al. [60] have also used multidimensional separation for analysis of normal and diseased heart tissue. Finding biomarkers for fast and accurate diagnosis of heart failure and screening for these is of extreme importance due to the fact that heart disease is the leading cause of mortality in the Western world.
Wagner et al. [61] presented a particularly interesting approach for multidimensional analysis of samples of peptides with molecular weights below 20 kDa. The authors report sample pre-fractionation with restricted access materials (RAM), which contain AEX or SCX functionalities. Samples were applied first to the RAM column, then to the IEX column, from which the samples were eluted onto one of the four RP separation columns. Elution peaks were collected and applied to the MALDI-TOF-MS for peptide mapping.
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MONOLITHIC COLUMNS FOR ANALYSIS OF PROTEOMIC SAMPLES |
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One of the bottlenecks for proteomics research is the sample throughput. Sample separation is time-consuming, often taking several hours per sample.
Development of stationary phases for HPLC has been taking place for more than 35 years, however, the field has not come to saturation, since the need for columns suitable for high-speed and high-resolution HPLC is enormous, especially in proteomics.
‘All separations in chromatography would benefit from high resolution, high column capacity, fast separations and low back pressures’ [62]. A very simple approach to come close to achieve these goals is to use monolithic columns for separations in proteomics [63].
For monolithic columns, the chromatographic bed consists of a single piece of porous polymer. Monolithic columns offer easy preparation and functionalization, enhanced mass transfer, high column efficiency, and they are more robust compared to pellicular HPLC columns. However, monolithic columns suffer from one very important limitation: they are very often and very easily overloaded. While this might not be a problem in conventional HPLC analysis, it becomes an overwhelming obstacle when a monolithic column is used as a separation column for the second dimension. Sample volumes directed from the first dimension must be reduced in order to benefit from the monolithic column and not overload it. One possible way to resolve these problems is to use serial linkage of these columns as described by Gray et al. [62]. If this type of linkage is applied for the second dimension of a separation system, the amount of sample from the first dimension on each of the monolithic columns becomes only a fraction of that which would be present on a single column.
Another application of monolithic columns is the use of online enzymatic reactors. Duan et al. [64] immobilized trypsin on a monolithic column and compared the digestion with an off-line in-solution digestion. The offline digestion took about 12 h, whereas a digestion of comparable efficiency on the monolithic column took about 30 s. This approach needs further tuning but it seems to be promising in terms of increasing the sample throughput.
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MINIATURIZATION |
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Another important aspect of future development is the miniaturization of instrumentation, primarily of the separation columns. There are many publications describing miniaturized chips and their use for separation of proteins and peptides. Initially, the use was not focused on sample infusion with ESI-MS [65, 66]. Later, the use of preformed silica tips to the separation device was published [67–69]. These chips suffered from high dead volumes and high chemical background in MS, originating from the glue for the tips. Furthermore, the majority of chip developments focused on electrophoresis, not on HPLC.
Yin et al. [70] reported the development of a new chip designed to use existing nano-HPLC hardware and MS. Fortier et al. [71] report the use of the nano-HPLC chip for RP and 2D separation of peptides obtained through tryptic digest of rat plasma.
Reichmuth et al. [72] demonstrated the use of a C18 side chain porous monolithic column with an integrated fluorescence detector for separation of peptides and proteins.
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ULTRA HIGH PRESSURE LIQUID CHROMATOGRAPHY |
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Pumps and columns used in ‘conventional’ HPLC operate in pressure ranges of up to 250 bar (separation columns) and 450 bar (pumps). This limits the use of new stationary phases with particle sizes of
2 µm. Decreasing the particle size by a factor of 2, from 3 to 1.5 µm, would increase the back-pressure 4-fold (pressure is inversely proportional to the square of the particle size). The use of small particles for the stationary phase would improve the peak shape and increase the column resolution. Wilson et al. [73] report the use of ultra performance chromatography (UPLC) for functional genomic discrimination of metabolic phenotypes. The results achieved with UPLC were significantly better in comparison to the results achieved with conventional HPLC. Another application recently published by Wu and Engen [74] reports the use of UPLC for hydrogen/deuterium exchange mass spectrometry. The authors compared deuterium loss observed with UPLC and HPLC and were able to improve on their results previously obtained with a normal HPLC system.
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CONCLUSIONS |
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The use of HPLC in proteomics is widely accepted for separation of proteins and peptides. This technique continues to develop through new separation phases and new instrumentation. Development of ICAT and monolithic columns as well as the development of new affinity column media like titanium and zinc have introduced new tools for selective sample cleanup and enrichment, and have therefore enabled the researcher to dig deeper into the proteome.
RP separation on packed columns remains the predominant type of sample separation. However, the use of monolithic columns with a C18 surface and built-in functionalities like IEX, RAM and affinity monolithic columns is developing rapidly.
‘Conventional’ nano-HPLC using flow rates of 200–300 nl/min to separate peptides on 75–100 µm ID columns still dominates the field. However, new emerging approaches like UPLC and miniaturization of complete separation systems on a chip will certainly change the profile of separation techniques for proteomics.
Certainly, enhancements in separation techniques must be followed by improvements in the field of MS, enabling it to keep pace with fast-developing separation technology.
Key Points
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Appendix |
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References on the web concerning HPLC instrumentation for proteomics:
- Agilent: http://www.agilent.com
- Dionex Corporation: http://www.dionex.com
- Eksigent: http://www.eksigent.com
- LC Packings—A Dionex Company: http://www.dionex.com
- Michrom: http://www.michrom.com
- Useful link for discussions and news from the proteomic society: http://www.abrf.org
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Acknowledgments |
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James Hutchins provided valuable help and interesting discussion. This work is supported by the Austrian Proteomics Platform (APP) within the Austrian Genome Research Program (GEN-AU) and by the Mitocheck project within the Sixth Framework Program of the European Commission.
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FOOTNOTES |
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Goran Mitulovi
is currently a postdoc with the Research Institute of Molecular Pathology in Vienna, Austria. He earned his PhD from the University of Vienna in 2001 and was research associate with LC Packings in Amsterdam from 2001 to 2004. His main area of work is (multidimensional) nano HPLC hyphenated with mass spectroscopy for proteomics applications. Karl Mechtler is the Head of Protein Chemistry Facility at the Research Institute of Molecular Pathology in Vienna. His main area of research is mass spectrometric analysis of peptides and proteins with focus on posttranslational modifications.
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References |
|---|
- Wilkins MR, Sanchez JC, Golley AA, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Genet Eng Rev 1996; 13:19–50.
- O’Farrel PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975; 250:4007–21.
[Abstract/Free Full Text] - Gorg A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000; 21:1037–53.[CrossRef][Web of Science][Medline]
- Mann M, Hendrickson RC, Pandey A. Analysis of proteins and proteomes by mass spectrometry. Anun Rev Biochem 2001; 70:437–73.
- Shevchenko A, Jensen ON, Podtelejnikov A, et al. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 1996; 93:14440–45.
[Abstract/Free Full Text] - Mitulovi G, Smoluch M, Chervet J-P, et al. An improved method for tracking and reducing the void volume in nano HPLC-MS with micro trapping columns. Anal Bioanal Chem 2003; 376:946–51.[CrossRef][Web of Science][Medline]
- Schaefer H, Chervet J-P, Bunse C, et al. A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects. Proteomics 2004; 4:2541–44.[CrossRef][Web of Science][Medline]
- Moore RE, Licklider L, Schumann D, et al. A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography/tandem mass spectrometry analysis of peptides and proteins. Anal Chem 1998; 70:4879–84.[Medline]
- Davis MT, Lee TD. Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer. J Am Soc Mass Spectrom 1998; 9:194–201.[CrossRef][Web of Science][Medline]
- Washburn M, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001; 19:242–47.[CrossRef][Web of Science][Medline]
- Wolters DA, Washburn MP, Yates JR III. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 2001; 73:5683–90.[Medline]
- VerBerkmoes NC, Bundy JL, Hauser L, et al. Integrating top-down and bottom-up mass spectrometric approaches for proteomic analysis of Shewanella oneidensis. J Proteome Res 2002; 1:239–52.[CrossRef][Web of Science][Medline]
- VerBerkmoes NC, Strader MB, Smiley RD, et al. Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase. Anal Biochem 2002; 305:68–81.[CrossRef][Web of Science][Medline]
- Belov ME, Gorshkov MV, Udseth HR, et al. Zeptomole-sensitivity electrospray ionization-fourier transform ion cyclotron resonance mass spectrometry of proteins. Anal Chem 2000; 72:2271–9.[Medline]
- Chervet J-P, Ursem M, Salzmann J-P. Instrumental requirements for nanoscale liquid chromatography. Anal Chem 1996; 68:1507–12.[CrossRef]
- Peng J, Gygi SP. Proteomics: the move to mixtures. J Mass Spectrom 2001; 36:1083–91.[CrossRef][Web of Science][Medline]
- Shen Y, Toli N, Zhao , et al. High-throughput proteomics using jhigh-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry. Anal Chem 2001; 73:3011–21.[Medline]
- Shen Y, Zhao R, Belov ME, et al. Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization fourier transform ion cyclotron resonance mass spectrometry for proteomics. Anal Chem 2001; 73:1766–75.[Medline]
- Shen Y, Moore R, Zhao R, et al. High-efficiency on-line coupling to 15–150-µm-i.d. Column liquid chromatography for proteomic analysis. Anal Chem 2003; 75:3596–605.[Medline]
- Quannzhou L, Tang K, Yang F, et al. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization – mass spectrometry. J Proteome Res 2006; 5:1091–7.[CrossRef][Web of Science][Medline]
- Rucevic M, Clifton J, Huang F, et al. Use of short monolithic columns for isolation of low abundance membrane proteins. J Chromatogr A 2006; 1123:199–204.
- Premstaller A, Oberacher H, Walcher H, et al. High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies. Anal Chem 2001; 73:2390–6.[Medline]
- Ishizuka N, Kobayashi H, Minakuchi H, et al. Monolithic silica columns for high-efficiency separations by high-performance liquid chromatography. J Chromatogr A 2002; 960:85–96.
- Vissers JP, Chervet J-P, Salzmann J-P. Sodium dodecyl sulphate removal from tryptic digest samples for on-line capillary liquid chromatography/electrospray mass spectrometry. J Mass Spectrom 1996; 31:1021–27.[CrossRef][Web of Science][Medline]
- van der Heeft E, ten Hove GJ, Herberts CA, et al. A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules. Anal Chem 1998; 70:3742–51.[Medline]
- Wassinger VC, Cordwell SJ, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 1995; 16:1090–4.[CrossRef][Web of Science][Medline]
- Wilkins MR, Pasquali C, Appel RD, et al. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Bio Technol 1996; 14:61–5.
- Gruber KA, Stein S, Brink L, et al. Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues. Proc Natl Acad Sci USA 1976; 73:1314–8.
[Abstract/Free Full Text] - O’Donovan C, Apweiler R, Bairoch A. The human proteomics initiative (HPI). Trends Biotechnol 2001; 19:178–81.[CrossRef][Web of Science][Medline]
- Hosoya K, Watabe Y, Kubo , et al. Novel surface-modification techniques for polymer-based separation media stimulus-responsive phenomena based on double polymeric selectors. J Chromatogr A 2004; 1030:237–46.
- Ohguro H, Palczewski K. Separation of phosphor- and non-phosphopeptides using reverse phase column chromatography. FEBS Letters 1995; 368:452–4.[CrossRef][Web of Science][Medline]
- Blacken GR, Gelb MH, Ture
ek F. Metal affinity capture tandem mass spectroscopy for the selective detection of phosphopeptides. Anal Chem 2006; 78:6065–73.[Medline] - Sano A, Nakamura H. Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal Sci 2004; 20:861–4.[CrossRef][Web of Science][Medline]
- Ikeguchi Y, Nakamura H. Fluorometric determination of phospholipids by high-performance liquid chromatography with postcolumn on-line hydrolysis under UV-Irradiation, phosphomolybdic acid and thiamine-thiochrome reactions. Anal Sci 1999; 15:229–32.[Medline]
- Sano A, Nakamura H. Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides. Anal Sci 2004; 20:565–6.[CrossRef][Web of Science][Medline]
- Pinkse MWH, Uitto PM, Hilhorst J, et al. Proceedings of 52nd ASMS Conference on Mass Spectrometry Allied TopicsNashville, TN 2004 WPU 400.
- Pinkse MWH, Uitto PM, Hilhorst J, et al. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 2004; 76:3935–43.[Medline]
- Larsen MR, Thingholm TE, Jensen ON, et al. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 2005; 4:873–86.
[Abstract/Free Full Text] - Kuroda I, Shintani Y, Motokawa M, et al. Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal Sci 2004; 20:1313–9.[CrossRef][Web of Science][Medline]
- Hofmann F, Dostmann W, Keilbach A, et al. Structure and physiological role of cGMP-dependent protein kinase. Biochim Biophys Acta 1992; 1135:51–60.[Medline]
- Schlosser A, Vanselow JT, Kramer A. Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Anal Chem 2005; 77:5243–50.[Medline]
- Meiring HD, van der Heeft E, ten Hove GJ, et al. Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis. J Sep Sci 2002; 25:557–68.[Medline]
- Andersson L, Porath J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 1986; 154:250–4.[CrossRef][Web of Science][Medline]
- Michael H, Hunt DF, Schabanowitz J, et al. Tandem mass spectrometry reveals that three photosystem II proteins of spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2-terminin. J Biol Chem 1988; 263:1123–30.
[Abstract/Free Full Text] - Ren D, Penner NA, Slentz BE, Regnier F. Histidine-rich peptide Selection and Quantification in Targeted Proteomics. J Proteome Res 2004; 3:37–45.[CrossRef][Web of Science][Medline]
- Ficcaro SB, McCleland ML, Stukkenberg PT, et al. Phosphoproteome analysis and its application to Saccharomyces cerevisiae. Nat Biotechnol 2002; 20:301–5.[CrossRef][Web of Science][Medline]
- Cantin GT, Venable JD, Cociorva D, et al. Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway. J Proteome Res 2006; 5:127–34.[CrossRef][Web of Science][Medline]
- Wang J, Zhang Y, Jiang H, et al. Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS. Proteomics 2006; 6:404–11.[CrossRef][Web of Science][Medline]
- Feuerstein I, Morandell S, Stecher G, et al. Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder. Proteomics 2005; 5:46–54.[CrossRef][Web of Science][Medline]
- Wolters DA, Washburn MP, Yates JR III. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 2001; 73:5683–90.[Medline]
- Mitulovi G, Stingl C, Smoluch M, et al. Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests. Proteomics 2004; 4:2545–57.[CrossRef][Web of Science][Medline]
- Le Bihan T, Duewel HS, Figeys D. On-line strong cation exchange µ-HPLC-ESI-MS/MS for protein identification and process optimization. J Am Soc Mass Spectrom 2003; 14:719–27.[CrossRef][Web of Science][Medline]
- Wagner Y, Sickman A, Meyer H, et al. Multidimensional nano-HPLC for analysis of protein complexes. J Am Soc Mass Spectrom 2003; 14:1003–11.[CrossRef][Web of Science][Medline]
- Ballif BA, Villen J, Beausoleil SA, et al. Phosphoproteomic Analysis of the Developing Mouse Brain. Mol Cell Proteomics 2004; 3:1093–101.
[Abstract/Free Full Text] - Winnik WM. Continuous pH/salt gradient and peptide score for strong cation exchange chromatography in 2D-nano-LC-MS/MS peptide identification for proteomics. Anal Chem 2005; 77:4994–8.
- Millea KM, Krull IS, Cohen SA, et al. Integration of multidimensional chromatographic protein separations with a combined top-down and bottom-up proteomic strategy. J Proteome Res 2006; 5:135–46.[CrossRef][Web of Science][Medline]
- Kelleher NL. Top-down proteomics. Anal Chem 2004; 76:197A–203A.[Medline]
- LeDuc RD, Taylor GK, Kim Y-B, et al. ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res 2004; 32:W340–5.
[Abstract/Free Full Text] - Hayter JR, Robertson DH, Gaskell SJ, et al. Proteome analysis of intact proteins in complex mixtures. Mol Cell Proteomics 2003; 2:85–95.
[Abstract/Free Full Text] - Kislinger T, Gramolini AO, MacLennan AO, et al. Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue. J Am Soc Mass Spectrom 2005; 16:1207–20.[CrossRef][Web of Science][Medline]
- Wagner K, Miliotis T, Marko-Varga G, et al. An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal Chem 2002; 74:809–20.[Medline]
- Gray MJ, Slonecker PJ, Dennis G, et al. A column capacity study of single, serial, and parallel linked rod monolithic high performance liquid chromatography columns. J Chromatogr A 2005; 1096:92–100.
- Tanaka N, Kobayashi H, Ishizuka N, et al. Monolithic silica columns for high-efficiency chromatographic separations. J Chromatogr A 2002; 965:35–49.
- Duan J, Sun L, Liang Z, et al. Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr A 2006; 1106:165–74.
- Xue Q, Foret F, Dunayevskiy YM, et al. Multichannel Microchip Electrospray Mass Spectrometry. Anal Chem 1997; 69:426–30.[Medline]
- Ramsey RS, Ramsey JM. Generating electrospray from microchip devices using electroosmotic pumping. Anal Chem 1997; 69:1174–8.[CrossRef]
- Zhang B, Liu H, Karger BL, et al. Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry. Anal Chem 1999; 71:3258–64.[Medline]
- Xiang F, Lin Y, Wen J, et al. An integrated microfabricated device for dual microdialysis and on-line ESI-ion trap mass spectrometry for analysis of complex biological samples. Anal Chem 1999; 71:1485–90.[Medline]
- Figeys D, Ning Y, Aebersold R. A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry. Anal Chem 1997; 69:3153–60.[Medline]
- Yin H, Killeen K, Brennen R, et al. Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip. Anal Chem 2005; 77:527–33.[Medline]
- Fortier M-H, Bonneil E, Goodley P, et al. Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs. Anal Chem 2005; 77:1631–40.[Medline]
- Reichmuth DS, Shepodd TJ, Kirby BJ. Microchip HPLC of peptides and proteins. Anal Chem 2005; 77:2997–3000.[Medline]
- Wilson ID, Nicholson JK, Castro-Perez J, et al. High resolution uUltra performance liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies. J Proteom Res 2005; 4:591–8.
- Wu Y, Engen JR, Hobbins WB. Ultra performance liquid chromatography (UPLC) further improves hydrogen/deuterium exchange mass spectrometry. J Am Soc Mass Spectrom 2006; 17:163–7.[CrossRef][Web of Science][Medline]
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