File Name: hplc of peptides and proteins methods and protocols .zip
In particular, HPLC in its various modes has become the central technique in the characterization of peptides and proteins and has, therefore, played a critical role in the rapid advances in the biological and biomedical sciences over the last 10 years. The enormous success of HPLC can be attributed to a number of inherent features associated with reproducibility, ease of selectivity manipulation, and generally high recoveries.
His main area of work is multidimensional nano HPLC hyphenated with mass spectroscopy for proteomics applications. His main area of research is mass spectrometric analysis of peptides and proteins with focus on posttranslational modifications. Due 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.
Proteomics, defined as the study of proteomes, started its growth in the mid s 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.
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. 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. Details of these methods have been reviewed in many articles and publications [ 4 , 5 ]. 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.
Belov et al. Here, the flow rates needed for operating such small ID columns decrease exponentially with column diameter. 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.
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. Samples for proteomic analysis are available in high amounts; however, the analytes are present in minute concentrations.
There are commercially available HPLC systems both with and without flow splitting. Briefly, in systems using flow splitting, a high pump flow rate of approx. 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.
Separation columns for proteomics research usually use the same stationary phase as for conventional HPLC. 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.
However, the trap columns must show high load capacity and low void volumes. Schaefer et al. 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.
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 ]. 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 for peptide separation [ 28 ].
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. 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 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. 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. 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.
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. 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. 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 ]. 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.
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. The affinity of titanium oxide TiO 2 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. 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. Here, automated column switching with MS detection of phosphopeptides was introduced and described. Phosphate buffer for elution of phosphopeptides was used at pH 7. 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.
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. 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 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.
Go back to menu. Correlation about 0. Petrilli et al. Higgins Columns Part Numbers and Prices. Importance of TFA Concentration for a reproducible peptide separation. Hao, et al. Proteome Res.
Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins e. Computational methods typically use computer programs to analyze proteins. However, many experimental methods e.
Almut Hesse, Michael G. Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes.
In the last couple of decades, considerable effort has been focused on developing methods for quantitative and qualitative proteome characterization. The method of choice in this characterization is mass spectrometry used in combination with sample separation. One of the most widely used separation techniques at the front end of a mass spectrometer is high performance liquid chromatography HPLC. A unique feature of HPLC is its specificity to the amino acid sequence of separated peptides and proteins.
It seems that you're in Germany. We have a dedicated site for Germany. High performance liquid chromatography HPLC plays a critical role today in both our understanding of biological processes and in the development of peptide and protein-based pharmaceuticals. In HPLC of Peptides and Proteins: Methods and Protocols, leading experts from academia and industry comprehensively describe how to successfully perform all the critical HPLC techniques needed for the analysis of peptides and proteins. The methods range from commonly used techniques to those for capillary to large-scale preparative isolation. The authors have also presented a number of specific applications as case studies to illustrate the analytical approaches to a particular separation or assay challenge, with examples drawn from contemporary fields in biochemistry and biotechnology. Each readily reproducible protocol contains background notes, step-by-step instructions, reagent and equipment lists, and tips on both troubleshooting and avoiding known pitfalls.
Metrics details. Nowadays, there is a growing interest in innovative and more efficient therapeutics—biopharmaceuticals, based on peptides or proteins. There are increased demands on quality control of such therapeutics. In this work, a modern advanced analytical method based on precolumn derivatization and reversed-phase ultra high-performance liquid chromatography in combination with single quadrupole mass spectrometer was developed for amino acid analysis in different protein samples—model sample of bovine serum albumin, sample of strong immunogenic protein keyhole limpet hemocyanin, and sample of drug etanercept present in commercially available biopharmaceutical Enbrel. The use of novel biologics, peptide therapeutics, therapeutic peptide conjugates, and therapeutic proteins continues to increase. There is a growing tendency to use them in treatment of several diseases such as various types of cancer, inflammation or neurodegeneration. Keyhole limpet hemocyanin KLH is an extracellular respiratory protein which is isolated from the Californian giant keyhole limpet Megathura crenulata.