METHODS IN MOLECULAR BIOLOGY TM Edited by Marie-Isabel Aguilar HPLC of Peptides and Proteins Volume 251 Methods and Protocols Edited by Marie-Isabel Aguilar Methods and Protocols HPLC of Peptides and Proteins METHODS IN MOLECULAR BIOLOGY TM 3 1 HPLC of Peptides and Proteins Basic Theory and Methodology Marie-Isabel Aguilar 1. Introduction High-performance liquid chromatography (HPLC) is now firmly established as the premier technique for the analysis and purification of a wide range of molecules. In particular, HPLC in its various modes has become the central tech- nique 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. The most significant feature is the excellent resolu- tion that can be achieved under a wide range of conditions for very closely related molecules, as well as structurally quite distinct molecules. This arises from the fact that all interactive modes of chromatography are based on recog- nition forces that can be subtly manipulated through changes in the elution con- ditions that are specific for the particular mode of chromatography. Peptides and proteins interact with the chromatographic surface in an orientation- specific manner, in which their retention time is determined by the molecular composition of specific contact regions. For larger polypeptides and proteins that adopt a significant degree of secondary and tertiary structure, the chro- matographic contact region comprises a small proportion of the total molecu- lar surface. Hence, the unique orientation of a peptide or protein at a particular stationary phase surface forms the basis of the exquisite selectivity that can be achieved with HPLC techniques. All biological processes depend on specific From: Methods in Molecular Biology, vol. 251, HPLC of Peptides and Proteins: Methods and Protocols Edited by: M I. Aguilar © Humana Press Inc., Totowa, NJ CH01,1-8,8pgs 10/30/03 7:00 PM Page 3 interactions between molecules and affinity chromatography exploits these spe- cific interactions to allow the purification of a biomolecule on the basis of its biological function or individual chemical structure. In contrast reversed phase HPLC, ion-exchange and hydrophobic interaction chromatography separate peptides and proteins on the basis of differences in surface hydrophobicity or surface charge. These techniques therefore allow the separation of complex mixtures whereas affinity chromatography normally results in the purification of one or a small number of closely related components of a mixture. Reversed-phase chromatography (RPC) is arguably the most commonly used mode of separation for peptides, although ion-exchange (IEC) and size exclu- sion (SEC) chromatography also find application. The three-dimensional struc- ture of proteins can be sensitive to the often harsh conditions employed in RPC, and as a consequence, RPC is employed less for the isolation of proteins where it is important to recover the protein in a biologically active form. IEC, SEC, and affinity chromatography are therefore the most commonly used modes for proteins, but RPC and hydrophobic interaction (HIC) chromatography are also employed. HPLC is extremely versatile for the isolation of peptides and proteins from a wide variety of synthetic or biological sources. The number of applications of HPLC in peptide and protein purification continue to expand at an extremely rapid rate. Solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides and proteins which need to be highly purified. The design of multidimensional purification schemes to achieve high levels of product purity further highlight the power of HPLC techniques in the analysis and isolation of peptide and proteins samples. The complexity of the mixture to be chromatographed depends on the nature of the source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large scale purification. The isolation of proteins from a biologi- cal cocktail however, often requires a combination of techniques to produce a homogenous sample. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule. The key factor that underpins the development of a successful separation protocol is the ability to manipulate the retention of the target mol- ecule so that it can be resolved from other contaminating components. This chapter thus provides an outline of the general theory of chromatography and the factors that control both the retention time and peakwidth of solutes under- going separation in terms of the parameters that control resolution. This infor- mation can then be used to understand the approaches used to perform 4 Aguilar CH01,1-8,8pgs 10/30/03 7:00 PM Page 4 separations with specific modes of chromatography as outlined in the remain- ing chapters in this book. 2. The Molecular Basis of Separation The separation of a mixture of peptides and proteins in interactive modes of chromatography arises from the differential adsorption of each solute accord- ing to their respective affinity for the immobilized stationary phase. Thus, when a particular molecule has a very high affinity for a specific stationary phase, i.e., when the equilibrium distribution coefficient K is high, then that solute is retained to a greater extent than another molecule with a lower affinity for the stationary phase. The degree and nature of the binding affinity is clearly depen- dent on the structure of the solute and the immobilized ligands. For example, in the case of RPC and HIC, binding is mediated predominantly through hydrophobic interactions between the solute and the immobilized n-alkyl lig- ands. In IEC, the binding is through electrostatic interactions, whereas in dif- ferent modes of affinity chromatography, binding involves a mixture of hydrophobic, electrostatic, and polar forces. In the case of size exclusion chro- matography, the differential movement along the column is a result of the extent to which each solute can permeate the porous structure of the stationary phase. An additional factor that influences the appearance and relative separation of a peak is the degree of bandbroadening of the solute band during migration through the column. Thus, as it moves down the column, the solute band broad- ens as a consequence of a number of factors including longitudinal diffusion, brownian motion, eddy diffusion, and mobile phase and stagnant phase mass transfer. These effects result in bandbroadening that generally increases with increasing residence time in the column. The resulting degree of separation or selectivity between constituent solutes in a mixture is thus a subtle interplay between the relative affinity of the molecules for the stationary phase and the degree of diffusive processes that occur during separation. 3. Retention and Bandwidth Relationships The time taken for a solute to pass though a chromatographic column is referred to as the retention time t r . This retention time is measured as the time taken by the solute, following injection, to emerge from the column and to be detected as illustrated in Fig. 1. In order to allow retention times to be com- pared to different columns or under different conditions, the retention time of a solute is normally compared with the retention time of a molecule which is not retained on the specific column of interest. This allows the unitless capac- ity factor k′ of a solute to be expressed in terms of the retention time t r , through the relationship HPLC of Peptides and Proteins 5 CH01,1-8,8pgs 10/30/03 7:00 PM Page 5 k′ = (t r – t o ) / t o (1) where t o is the retention time of a nonretained solute. The capacity factor k′ can also be defined as the ratio n s /n m where n s and n m are the number of moles of solute in the stationary phase and mobile phase respectively as follows: k′ = n s / n m (2) or alternatively as k′ = [X] s V s / [X] m V m (3) where [X] s and [X] m refer to the concentrations of the solute in the stationary and mobile phases, respectively, and V s and V m are the corresponding volumes of the stationary and mobile phases. Since the ratio [X] s / [X] m is the equilibrium dis- tribution coefficient K and the ratio V s / V m defines the phase ratio Φ of the chro- matographic system, the capacity factor can also be expressed as follows: k′ = Φ[X] s / [X] m (4) or k′ = ΦK (5) 6 Aguilar Fig. 1. Diagram of the retention parameters that describe a chromatographic sepa- ration. The retention time of a nonretained solute is denoted by t 0 , while the retention times of two retained solutes, 1 and 2, are given by t r,1 and t r,2 . The corresponding peak- widths for solutes 1 and 2 are denoted σ,1 and σ,2, and together with the retention times are they used to detrmine the resolution of the separation according to Eq. 9. CH01,1-8,8pgs 10/30/03 7:00 PM Page 6 Equation 5 thus formerly describes the direct thermodynamic relationship between the retention of a peptide or protein and its affinity for the stationary phase material. The practical significance of k′ can be related to the selectivity parameter α, defined as the ratio of the capacity factors of two adjacent peaks as follows: α = k′ i / k′ j (6) which allows the definition of a chromatographic elution window in which retention times can be manipulated to maximise the separation of components within a mixture. Clearly, the aim is to obtain as high a value of α as possible, which reflects a high degree of separation between two peaks. The second factor involved in defining the quality of a separation is the peak width σ t . The degree of peak broadening is directly related to the efficiency of the column and can be expressed in terms of the number of theoretical plates N as follows: N = (t r ) 2 / σ r 2 . (7) N can also be expressed in terms of the reduced plate height equivalent h, the column length L, and the particle diameter of the stationary phase material d p ,as N = hL / d p . (8) The resolution R s between two components of a mixture, therefore, depends on both selectivity and bandwidth according to R s = 1 / 4 √N (α – 1)[1 / (1 + k′)]. (9) This equation describes the relationship between the quality of a separation and the relative retention, selectivity, and the bandwidth. It also provides the formal basis upon which resolution can be manipulated to achieve a particular level of separation. Thus, when faced with an unsatisfactory separation, the aim is to improve resolution by one of three possible strategies. The first is to increase α as previously and the second, but related, approach is to vary k′ within a defined range normally 1 < k′ < 10 through variation in the experimental elu- tion conditions such as solvent strength, separation time, or nature of the immo- bilized ligand. Third, one can increase N,for example, by using very small particles in microbore or narrow bore columns. An appreciation of the factors that control the resolution of peptides and pro- teins in interactive modes of chromatography can assist in the development and manipulation of separation protocols to obtain the desired separation. The opti- mization of high-resolution separations of peptides and proteins involves the separation of sample components through manipulation of both retention times and solute peak shape. For example, inspection of the schematic separation shown in Fig. 1 demonstrates baseline separation between the two components HPLC of Peptides and Proteins 7 CH01,1-8,8pgs 10/30/03 7:00 PM Page 7 which corresponds to a high value of both selectivity α, and resolution. A sce- nario can be envisaged where it may be desirable to decrease the retention times of the solutes to allow more rapid analysis times. However, resolution may be sacrificed and the final separation conditions are often likely to be a tradeoff between rate of analysis and quality of separation. An enormous range of different separation techniques are available for pep- tide and protein analysis. The challenge facing the scientist who wishes to ana- lyze and/or purify their peptide or protein sample is the selection of the initial separation conditions and subsequent optimisation of the appropriate experi- mental parameters. The following chapters thus provide a practical guide to per- forming peptide and protein analyses under a range of different separation modes. In addition, the reader is guided through the experimental options avail- able to achieve a high-resolution separation of a peptide or protein mixture, an exercise which is underpinned by the theoretical relationships provided in this chapter. 8 Aguilar CH01,1-8,8pgs 10/30/03 7:00 PM Page 8 9 2 Reversed-Phase High-Performance Liquid Chromatography Marie-Isabel Aguilar 1. Introduction Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity. The sepa- ration depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the station- ary phase, i.e., the sorbent. A schematic diagram showing the binding of a pep- tide or a protein to a reversed-phase surface is shown in Fig. 1. The solute mixture is initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by the addition of organic solvent to the mobile phase. Elution can proceed either by isocratic conditions where the concentration of organic solvent is constant, or by gradient elution whereby the amount of organic solvent is increased over a period of time. The solutes are, therefore, eluted in order of increasing molecular hydrophobicity. RP-HPLC is a very powerful technique for the analysis of peptides and proteins because of a number of fac- tors that include: (1) the excellent resolution that can be achieved under a wide range of chromatographic conditions for very closely related molecules as well as structurally quite distinct molecules; (2) the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase characteristics; (3) the generally high recoveries and, hence, high pro- ductivity; and (4) the excellent reproducibility of repetitive separations carried out over a long period of time, which is caused partly by the stability of the sor- bent materials under a wide range of mobile phase conditions (1,2). However, RP-HPLC can cause the irreversible denaturation of protein samples thereby reducing the potential recovery of material in a biologically active form. From: Methods in Molecular Biology, vol. 251, HPLC of Peptides and Proteins: Methods and Protocols Edited by: M I. Aguilar © Humana Press Inc., Totowa, NJ CH02,9-22,14pgs 10/30/03 6:59 PM Page 9 The RP-HPLC experimental system for the analysis of peptides and proteins usually consists of an n-alkylsilica-based sorbent from which the solutes are eluted with gradients of increasing concentrations of organic solvent such as ace- tonitrile containing an ionic modifier such as trifluoroacetic acid (TFA) (1,2). Complex mixtures of peptides and proteins can be routinely separated and low picomolar—femtomolar amounts of material can be collected for further charac- terization. Separations can be easily manipulated by changing the gradient slope, the operating temperature, the ionic modifier, or the organic solvent composition. The extensive use of RP-HPLC for the purification of peptides, small polypep- tides with molecular weights up to 10,000, and related compounds of pharma- ceutical interest has not been replicated to the same extent for larger polypeptides 10 Aguilar Fig. 1. Schematic representation of the binding of (A) a peptide and (B) a protein, to an RP-HPLC silica-based sorbent. The peptide or protein interacts with the immo- bilized hydrophobic ligands through the hydrophobic chromatographic contact region. CH02,9-22,14pgs 10/30/03 6:59 PM Page 10 (molecular mass > 10 KDa) and globular proteins. The combination of the traditionally used acidic buffering systems and the hydrophobicity of the n-alkylsilica supports which can result in low mass yields or the loss of biolog- ical activity of larger polypeptides and proteins have often discouraged practi- tioners from using RP-HPLC methods for large-scale protein separations. The loss of enzymatic activity, the formation of multiple peaks for compositionally pure samples, and poor yields of protein can all be attributed to the denaturation of protein solutes during the separation process using RP-HPLC (3–6). RP-HPLC is extremely versatile for the isolation of peptides and proteins from a wide variety of synthetic or biological sources and is used for both ana- lytical and preparative applications (1–2, see also Chs. 10–21). Analytical applications range from the assessment of purity of peptides following solid- phase peptide synthesis (see Ch. 14), to the analysis of tryptic maps of proteins. Preparative RP-HPLC is also used for the micropurification of protein fragments for sequencing to large-scale purification of synthetic peptides and recombi- nant proteins. The complexity of the mixture to be chromatographed will depend on the nature of the source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RP-HPLC is generally employed both for the initial analysis and the final large-scale purification. The purifica- tion of synthetic peptides usually involves an initial separation on an analyti- cal scale to assess the complexity of the mixture followed by large-scale purification and collection of the target product. A sample of the purified mate- rial can then be subjected to RP-HPLC analysis under the same or different elu- tion conditions to check for purity. The isolation of proteins from a biological cocktail derived from a tissue extract or biological fluid for example, often requires a combination of techniques to produce a homogenous sample. HPLC techniques are then introduced at the later stages following initial precipitation, clarification, and preliminary separations using soft gels. The challenge facing the scientist who wishes to analyze and/or purify their peptide or protein sample by RP-HPLC is the selection of the initial separation conditions and subsequent optimization of the appropriate experimental para- meters. This chapter describes a standard method that can be used as an initial procedure for the RP-HPLC analysis of a peptide sample, and then different experimental options available to achieve a high-resolution separation of a pep- tide or protein mixture using RP-HPLC are outlined in Subheading 4. 2. Materials 2.1. Chemicals 1. Acetonitrile (CH 3 CN), HPLC grade. 2. Milli-Q water. 3. Trifluoroacetic acid (TFA). RP-HPLC 11 CH02,9-22,14pgs 10/30/03 6:59 PM Page 11 [...]... DEAE-2SW prepacked HPLC column (see Table 3) has a pore size of 125 Å and an exclusion limit of 10,000 Daltons, whereas the TSK DEAE5PW HPLC column has a pore size of 1000 Å and an exclusion limit of 1,000,000 Daltons Invariably, the choice of IEX support will involve some level of compromise between the main factors that influence resolution (particle type and size, porosity) and capacity and cost Small... However, if the efficient recovery of both mass and biological activity is of paramount importance, the use of elevated temperatures is not an option 9 The choice of gradient conditions will depend on the nature of the molecules of interest The influence of gradient time on the separation of a series of tryptic peptides proteins is shown in Fig 6 (1) Generally the use of longer gradient times provides... molecular basis of the effect of ligand structure is not fully understood, a number of factors including the relative hydrophobicity and ligand chain length, flexibility, and the degree of exposure of surface silanols all play a role in the retention process An example of the effect of chain length on peptide separations can be seen in Fig 3 (1) It can be seen that the peaks labeled T3 and T13 are fully... isocratic elution of peptides and proteins can rarely be achieved as the experimental window of solvent concentration required for their elution is very narrow Mixtures of peptides and proteins are therefore routinely eluted by the application of a gradient of increasing organic solvent concentration The three most commonly employed organic solvents in RP -HPLC are acetonitrile, methanol, and 2-propanol,... examples of the use of nonporous particles of smaller diameter (18) For preparative scale separations, 10–20 µm particles are utilized The pore size of RP -HPLC sorbents is also an important factor that must be considered For peptides, the pore size generally ranges between 100–300 Å depending on the size of the peptides Porous materials of ≥300 Å pore size are necessary for the separation of proteins,. .. Enzymol 271, 3–50 3 Purcell, A W., Aguilar, M I., and Hearn, M T W (1995) Conformational effects in the RP -HPLC of polypeptides II: The role of insulin A and B chains in the chromatographic behaviour of insulin J Chromatogr 711, 71–79 4 Richards, K L., Aguilar, M I., and Hearn, M T W (1994) The effect of protein conformation on experimental bandwidths in RP -HPLC J Chromatogr 676, 33–41 5 Oroszlan, P., Wicar,... HPLC of Biological Macromolecules: Methods and Applications (Gooding, K M and Regnier, F E., eds.), Dekker, New York, pp 3–24 8 Henry, M (1991) Design requirements of silica-based matrices for biopolymer chromatography J Chromatogr 544, 413–443 CH02,9-22,14pgs 10/30/03 RP -HPLC 7:00 PM Page 21 21 9 Zhou, N E., Mant, C T., Kirkland J J., and Hodges R S (1991) Comparison of silica-based cyanopropyl and. .. for the isolation of more hydrophobic proteins such as membrane proteins (24, see Chapter 22) One of the most powerful characteristics of RP -HPLC is the ability to manipulate solute retention and resolution through changes in the composition of the mobile phase In RP -HPLC, peptide and protein retention is a result of multisite interactions with the ligands The practical consequence of this is that high... With microbore columns (1–2 mm id) flow rates of 50–250 µL/min are used, whereas for capillary columns of 0.2–0.4 mm id, flow rates of 1–4 µL/min are applied At the preparative end of the scale with columns of 10–20 mm id, flow rates ranging between 5–20 mL/min are required Detection of peptides and proteins in RP -HPLC, generally involves detection between 210 and 220 nm, which is specific for the peptide... for the analysis of lysozyme on a C18 material packed into columns of 4.6, 2.1, and 0.3 mm id (17) 3 The geometry of the particle in terms of the particle diameter and pore size, is also an important feature of the packing material Improved resolution can be achieved by decreasing the particle diameter and the most commonly used range of particle diameters for analytical scale RP -HPLC is 3–5 µm There . METHODS IN MOLECULAR BIOLOGY TM Edited by Marie-Isabel Aguilar HPLC of Peptides and Proteins Volume 251 Methods and Protocols Edited by Marie-Isabel Aguilar Methods and Protocols HPLC of. can be achieved with HPLC techniques. All biological processes depend on specific From: Methods in Molecular Biology, vol. 251, HPLC of Peptides and Proteins: Methods and Protocols Edited by:. the isolation of peptides and proteins from a wide variety of synthetic or biological sources. The number of applications of HPLC in peptide and protein purification continue to expand at an extremely rapid