606 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Cu ++ + Cu ++ Cu ++ matrix ligand Initial column Step 1. load metal Step 2. Bind protein (P) 7 < pH < 8 + P P Step 3 Desorb protein Cu ++ + P + EDTA Step 4 Regenerate column + Cu ++ EDTA chelator 4 < pH < 5 Figure 13.19 Steps in the use of IMAC. Adapted from [56]. amino-acid side chains in the protein can form coordination complexes with metals, so IMAC is a general method for protein separation. The primary interaction in IMAC is with the imidazole group of histidine in its unprotonated form [58]; the strength of metal binding by different amino-acid groups in the protein molecule decreases in the following order: his > trp > tyr > phe > arg ∼ met ∼ gly Cysteine residues can bind metals, but they may not be available on the protein surface in the reduced state, since they readily oxidize in the presence of metal ions [59]. Cysteine-containing proteins may therefore require a reducing environment (addition of 2-mercaptoethanol or dithiothreitol) in order to maintain the cysteine residues in their active (–SH) form. Aromatic residues can contribute indirectly to retention, by enhancing the binding of neighboring histidines [59]. The dominance of histidine binding to IMAC columns has been exploited by genetically engineering polyhistidyl sequences into target proteins for ease of purification. After preferential binding, elution, and recovery of the target protein, the polyhistidine sequence can be cleaved by means of carboxypeptidase A [59]. Phosphoproteins and phosphopeptides bind selectively to IMAC columns chelated with Fe +3 and Ga +3 , and IMAC has become a key tool in characterizing the phosphoproteome [60]. 13.4 SEPARATION OF PEPTIDES AND PROTEINS 607 The high capacity of IMAC columns and the high recovery of protein mass and activity make this technique useful for preparative and process-scale chro- matography. For protein purification, IMAC compares favorably with affinity chromatography in terms of binding strength and capacity and has the advan- tages of stability over a wide range of conditions and use-cycles, relatively mild elution conditions, and modest cost. In process chromatography, IMAC is best used as the initial step in a process sequence so that downstream steps such as IEC or hydrophobic interaction chromatography can eliminate any metals leached from the column during IMAC elution [37] (oxidation of protein residues can be catalyzed by metal ions). Selectivity in IMAC can be controlled by the choice of: • chelating ligand (i.e., the column) • immobilized metal ion • mobile-phase pH and ionic strength • any mobile-phase additives used to enhance binding or elute proteins Chelating Ligand. The chelating ligand–metal complex must be strong enough to be stable, but must also have metal-coordination sites available in order to bind the protein. The metal should be easily removed with a chelating agent such as EDTA, in order to allow column regeneration and conversion to another metallic form. The most common chelating groups used in IMAC are iminodiacetic acid (IDA) and tricarboxymethylethylenediamine (TED), which form tridentate and pentadentate metal complexes, respectively (Fig. 13.20). Although the pentadentate TED has stronger metal affinity, the tridentate IDA-metal complex leaves more metal coordination sites free for solute binding so that IDA-metal columns can exhibit higher protein affinity [55].The metal complex stability for IDA on an agarose support [56] is Cu 2+ > Ni 2+ > Zn 2+ ≥ Co 2+ > Fe 2+  Ca 2+ The corresponding affinity of TED for metals is Fe 3+ > Cu 2+ > Ni 2+ > Zn 2+ ∼ Co 2+ > Fe 2+ > Ca 2+ CH 2 CH 2 CH 2 CH 2 CH 2 -CO CH 2 -CO CH 2 -CO OH 2 OH 2 N O Me O Iminodiaceticacid (IDA) tricarboxymethylenediamine (TED) N CO O Me O N OH 2 O Figure 13.20 Structure of two common IMAC ligands. Adapted from [55]. 608 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Metal Ion. The selectivity of protein binding in IMAC depends primarily on the type of metal that is complexed with the chelating ligand. Protein–metal chelate interactions include coulombic and coordination bonding, while hydrophobic interactions may occur at high salt concentrations [55]. The most popular metals for IMAC are Cu 2+ ,Ni 2+ ,andZn 2+ . This popularity probably reflects the strong affinity of these metals for both the IMAC ligands and for proteins. The affinity of Cu 2+ for imidazole is 15-fold greater than Ni 2+ , which is 3-fold greater than Zn 2+ or Co 2+ [57]. The optimum pH and binding conditions are specific for a given protein–metal ion pair so they must be determined experimentally. Protein Binding and Elution. In addition to metal ion complexation, IMAC columns have the potential for ion-exchange, ion-exclusion, and hydrophobic inter- actions. The initial mobile phase for protein binding and the final mobile phase for elution can be designed to minimize or exploit these effects. Metal-free chelating groups function as cation-exchange sites and can interact with cationic groups on proteins (for an ionic strength < 0.1M). High concentrations of salt (≥ 0.5 M) suppress ion-exchange interactions but promote hydrophobic interactions with the chelating group or its linker. An intermediate ionic strength can suppress ion-exchange interactions and reduce the risk of protein aggregation, particularly for antibodies [37]. A general observation is that the retention of acidic proteins tends to increase with salt concentration, while the retention of basic proteins ini- tially decreases, then increases with increasing salt concentration [55]. Each of the latter separation conditions can be varied in order to optimize separation selectivity. Binding and elution is strongly affected by mobile-phase pH. Proteins bind most strongly above the pK a of the histidyl imidazole group (pK a ≈ 7), and binding strength is diminished as the pH drops below this value (and the histidine group becomes more ionized). Therefore a common elution strategy is to bind proteins at pH values between 7 and 8, followed by elution with a step or gradient to pH values between 4 and 5, in order to convert histidine residues to the ionized form. An alternative elution strategy is the use of a displacing agent such as imidazole, histamine, histidine, glycine, or ammonia. The first three agents are equivalent in eluting strength, and generally stronger than the latter two. Displacing agents of increasing strength can be introduced in sequence to elute weakly retained proteins first, followed by strongly retained species [61]. Proteins can also be resolved by using a concentration gradient of a single displacing agent. Like other separation techniques based on electrostatic interactions, IMAC ligands probe surface groups on the protein. Therefore conditions that alter or disrupt protein conformation can change selectivity in IMAC. The example in Figure 13.21 shows changes in retention and elution order caused by the addition of methanol to the mobile phase possibly reflecting changes in protein conformation). Two proteins differing by only a single, surface histine residue can be resolved by IMAC. 13.4.3 Hydrophobic Interaction Chromatography (HIC) Hydrophobic interaction chromatography (HIC) was first described by Tiselius [63] in the late 1940s, and later characterized by Porath [64] and Gelotte [65]. Since the 1960s, HIC has been widely used for protein separations with carbohydrate-based 13.4 SEPARATION OF PEPTIDES AND PROTEINS 609 5101520 % MeOH 15 10 5 0 k CYT LAC A LYS CHY Figure 13.21 Effect of added methanol on IMAC retention. Column: Fe (III)–IDA silica. Mobile phase: varying %-methanol in 25 mM phosphate (pH 6) + 0.15M ammonium sulfate. CYT, cytochrome C; LYS, lysozyme; LAC A, β-lactoglobulin A; CHY, chymotrypsinogen A. Adapted from [62]. packings, and more recently with high-performance microparticulate supports [66, 67]. The principle of HIC is based on the interaction of proteins with mildly hydrophobic ligands in the presence of high concentrations of salt. Proteins largely maintain their conformation under these conditions, and retention results from the interaction of hydrophobic patches on the protein surface with the column ligand (‘‘salting out’’). HIC retention is unusual in that it is an entropy-driven process ([68] and see below), and uses a reverse gradient (from high- to low-salt concentration). HIC is a gentle technique, with proteins eluting in their native conformation without loss of biological activity. It is therefore widely used for the preparative isolation of proteins in laboratory scale-up to process-scale applications. Although HIC and RPC share a retention mechanism based on hydrophobic interactions, the selectivity can be markedly different. The harsh conditions of RPC promote protein denaturation and exposure of internal hydrophobic residues, whereas retention in HIC only involves residues at the protein surface. A very different separation selectivity can therefore be expected. 13.4.3.1 Supports and Ligands for HIC Both silica- and polymer-based supports are used for HIC, with pore sizes that are large enough to allow penetration of the protein. The support is typically covered with a polymeric, hydrophilic coating (or linker groups) in order to provide a wettable, noninteractive surface; hydrophobic ligands are then attached to the polymer or linker. The ligands are typically short-chain alkyl or phenyl groups, and retention increases with ligand length ([70, 71]; see the examples of Figure 13.22 for three different proteins). Ligands of 1 to 3 carbons in length promote retention at high salt and release at low salt. Longer ligands can cause excessive retention, as well as induce conformational changes. 610 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS ribonuclease lysozyme chymotrypsinogen A 40 30 20 10 C 4 -C 3 OH C 1 C 3 C 2 Retention time (min) Figure 13.22 Effect of HIC ligand length on protein retention (–C 3 OH, hydroxypropyl; C 1 , methyl; etc.). Adapted from [69]. It should be noted that IEC and IMAC columns can each display HIC behavior under high-salt conditions, due to the hydrophobic contributions of spacer and cross-linking groups [68]. The latter columns can also exhibit multimodal retention behavior, depending on the operating conditions. When IEC and IMAC columns are used in a high-salt, HIC mode, the selectivity may be different from a conventional HIC column due to the contribution of non-HIC retention mechanisms. 13.4.3.2 Other Conditions The selectivity and retention in HIC separation is influenced by: • choice of antichaotropic salt and its concentration • mobile-phase pH • mobile-phase additives • temperature Antichaotropic Salt. The primary consideration in designing a HIC gradient is the selection of the salt and its concentrations at the beginning and end of the gradient. The ability of salts to promote retention in HIC parallels their effectiveness in protein precipitation as given by the Hofmeister salting-out series (Table 13.6); however, ammonium sulfate is the most widely used salt for HIC. Changing the salt type as well as its concentration can provide an opportunity for varying both retention and selectivity—but keep in mind the solubility and purity of the salt. Protein binding is achieved with an initial concentration of 1.5 to 3.0M salt; a reverse gradient to a lower salt concentration (or neat buffer) is then used for elution. For 13.4 SEPARATION OF PEPTIDES AND PROTEINS 611 log k 1.0 0.5 0.0 −0.5 −0.5 0.8 1.0 1.2 1.4 1.6 1.8 Salt concentration (M) pH = 8.0 7.0 6.0 pH = 8.0 7.0 6.0 log k 1.0 0.5 0.0 0.8 1.0 1.2 1.4 1.6 1.8 Salt concentration (M) Ring-necked pheasant Hen egg white (a) (b) Figure 13.23 Effect of pH on HIC retention of two different avian lysozymes (ring-necked pheasant, hen egg white) with varying ammonium sulfate molality. Adapted from [72]. large-scale preparative applications, the use of ammonium sulfate under alkaline conditions must be approached with caution, as free ammonia can be liberated [37]. In such cases potassium sulfate is an acceptable substitute. Mobile-Phase pH. This can affect selectivity if acidic or basic amino acids are located within the hydrophobic contact area. This is illustrated by the effect of histidine ionization state on the retention of avian lysozymes in HIC [72]. Ring-necked pheasant lysozyme has histidine residues in the contact region, and retention changes (Fig. 13.23a) as pH is varied across the pK a of the imidazole group (pK a = 6). Hen lysozyme, which has no histidines in the contact region, displays little pH-dependence of retention over the same pH range (Fig. 13.23b). A change in mobile-phase pH can therefore be used to separate these two protein variants. Additives. Retention in HIC is based on hydrophobic interaction, and it therefore should be affected by the addition of surfactants to the mobile phase (which 612 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS can bind to both the column and the protein, thereby reducing the hydrophobicity of each). Thus the addition of nonionic and zwitterionic surfactants reduces protein retention [68, 73]. Inclusion of surfactants is particularly useful in HIC separations of very hydrophobic species such as integral membrane proteins, which may require surfactants for solubilization. The addition of organic solvent to the mobile phase should also reduce protein retention, but this can be problematic for HIC. Organic solvents can induce conformational changes in the protein, and their use under conditions of high salt also introduces the risk of protein precipitation. Temperature. HIC retention is entropy driven and therefore increases with temperature—the opposite of the usual effect of temperature on retention. This effect is enhanced by conformational changes at higher temperatures that make internal hydrophobic residues available for increased interaction. Conformational effects can be recognized by peak broadening at temperatures intermediate between native and denaturing conditions [74], for example, between 10 and 35 ◦ Cin Figure 13.24. 0 5 10 15 20 25 30 10°C 25°C 32°C 35°C 40°C 50°C Figure 13.24 Effect of temperature on the HIC elution behavior of Ca 2+ -depleted α-lactalbumin. See text for details. Reprinted from [72] with permission. 13.4 SEPARATION OF PEPTIDES AND PROTEINS 613 13.4.4 Hydrophilic Interaction Chromatography (HILIC) Water-soluble solutes that are very hydrophilic and uncharged present the chro- matographer with a challenge, as they are poorly retained in RPC and unretained by IEC. Hydrophilic interaction chromatography (HILIC; Section 8.6; [75, 76]) provides a solution to this problem. In this technique a polar stationary phase is used with an aqueous-organic mobile phase. In contrast to RPC, the aqueous component of the mobile phase (e.g., water or buffer) serves as the strong solvent and the organic component (usually acetonitrile) is now the weak solvent; that is, retention increases as %-organic increases (Fig. 13.25). Note also that retention increases for more polar solutes in Figure 13.25 (Arg [most polar] > p-Ser > Leu [least polar])—again the opposite of RPC. HILIC can be considered as a variant of normal-phase chromatography (NPC; Chapter 8). In addition to achieving reasonable retention and separation of hydrophilic water-soluble analytes, HILIC has two other advantages. First, mobile phases with > 50% acetonitrile are less viscous, which means lower pressures and higher plate numbers. Second, these organic-rich mobile phases are ideal for efficient desolvation in electrospray-ionization LC-MS. 13.4.4.1 Stationary Phases for HILIC A variety of stationary phases have been used for HILIC [76], including underiva- tized silica [77], aminopropyl silica [78], amide silica [79], diol silica [80], sulfonated polystyrene-divinylbenzene [81], and poly(2-hydroxyethyl aspartamide) [75]. The use of bare silica as the stationary phase eliminates the problem of ligand bleed in LC-MS, which can occur when bonded phases are used. While the ionization Arg p-Ser Leu 020406080 % ACN 8 6 4 2 0 k Figure 13.25 Retention behavior of amino acids in hydrophilic interaction chromatography (HILIC). Conditions: cation-exchange column (PolySulfoethyl A) used in HILIC mode; mobile phase, 25-mM TEAP (pH-5.0) with acetonitrile as indicated. Adapted from [75]. 614 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS of silanols at pH ≥ 7 can introduce ion-exchange interactions and complicate the interpretation of the separation, the use of a mobile-phase pH < 7 avoids this problem. Alternatively, a bonded-phase HILIC column can be used; for example, silica-based amide columns have been used successfully for peptide separations [79]. Silica-based diol columns most closely approach the behavior of underiva- tized silica for HILIC separations, and minimize the problem of active silanols. Poly(2-hydroxyethyl aspartamide) is a stationary phase that was designed expressly for HILIC [77]; it is prepared by incorporating ethanolamine into a coating of polysuccinimide covalently bonded to silica. The resulting polyaspartamide coat- ing is a derivative of asparagine, the most hydrophilic neutral amino acid. Highly sulfonated polystyrene-divinylbenzene columns can be operated in a HILIC mode using acetonitrile-water eluents [81], with the advantage of stability provided by the polymeric support. However, these columns also function as ion-exchangers. Bare-silica and amide-silica columns were most often used for HILIC at the time this book was written. 13.4.4.2 Mobile Phases for HILIC Typical mobile phases for HILIC are mixtures of water (or aqueous buffer) and acetonitrile, with water content ranging from 5 to 40% [75]. Commonly used buffering agents are ammonium salts of formic or acetic acid, which are soluble in acetonitrile. These salts are also volatile for compatibility with electrospray ionization–mass spectrometry. The triethylamine salt of trifluoroacetic acid has also been recommended. Although the latter salt is volatile, however, TFA is notorious for causing suppression in electrospray ionization [17]. 13.4.4.3 Application of HILIC to Peptides and Proteins HILIC has been used successfully to separate peptides, and it exhibits a unique selec- tivity for peptides with hydrophilic post-translational modifications. Glycopeptide sequences with variations in glycan structure have been separated on an amide-silica column [82, 83], while a poly(2-hydroxyethyl aspartamide) column has been suc- cessful for the selective isolation of phosphopeptides [84]. The complementarity of HILIC and RPC was demonstrated in a study of the behavior of amphipathic α-helical peptides possessing hydrophobic and hydrophilic faces [85]. Substitutions in the hydrophilic face were shown to have little effect on RPC retention but to cause a change in relative retention in HILIC. Conversely, substitutions in the hydrophobic face affected RPC retention but not HILIC retention. This study also confirmed that different chromatographic contact areas are operative in the two modes. HILIC has not been widely used for proteins, due to the limited solubility of proteins in high concentrations of organic solvents. The appearance of multiple peaks for single proteins during HILIC has been reported [86, 87] and attributed to on-column denaturation. However, HILIC has been used successfully for the separation of histones (a family of highly basic DNA-binding proteins), including acetylated [88], phosphorylated [89], and methylated [90] histone variants. 13.4.4.4 Electrostatic-Repulsion Hydrophilic-Interaction Chromatography (ERLIC) This is a variation of HILIC that employs an ion-exchange column operated with a predominantly organic mobile phase [91]. With ERLIC, solutes can be retained 13.4 SEPARATION OF PEPTIDES AND PROTEINS 615 via hydrophilic interaction (HILIC mode), even if they have the same charge as the stationary phase. As a result it is possible to simultaneously separate mixtures of acids and bases that might otherwise be difficult to separate by either HILIC or IEC. The ERLIC technique can be regarded as possessing both hydrophilic-interaction and ion-exchange selectivity. The important feature of this combination is the independence of hydrophilic and electrostatic effects, allowing their independent manipulation. This is illustrated in Figure 13.26 by a comparison of HILIC and ERLIC separations of a mixture of acidic, neutral and basic peptides. In the HILIC separation (Fig. 13.26a) with a neutral, hydrophilic column, a concentration of acetonitrile (%-ACN) that provides suitable retention of basic peptides yields inadequate retention for acidic and neutral peptides. In the ERLIC separation of Figure 13.26b (performed with a weak anion-exchange column at low pH), electrostatic repulsion of the basic peptides reduces their retention, allowing %-ACN to be increased so that all peptides are retained and resolved. ERLIC has been used for the enrichment and separation of phosphopep- tides from a tryptic digest of HeLa cell proteins [92]. Under conventional weak acidic peptide basic peptide 0 20406080100 (min) ERLIC HILIC (a) (b) basic peptides Figure 13.26 Separation of peptide standards by HILIC (a) compared with ERLIC (b). HILIC conditions: PolyHydroxyethyl A column (PolyLC, Columbia, Maryland); mobile phase, 20-mM Na-MePO 4 (pH-2.0) +63% acetonitrile. ERLIC conditions: column, PolyWAX LP; mobile phase, 20-mM Na-MePO 4 (pH-2.0) +70% acetonitrile. Adapted from [91]. . environment (addition of 2-mercaptoethanol or dithiothreitol) in order to maintain the cysteine residues in their active (–SH) form. Aromatic residues can contribute indirectly to retention, by enhancing. elution strategy is to bind proteins at pH values between 7 and 8, followed by elution with a step or gradient to pH values between 4 and 5, in order to convert histidine residues to the ionized form. An. sulfate. CYT, cytochrome C; LYS, lysozyme; LAC A, β-lactoglobulin A; CHY, chymotrypsinogen A. Adapted from [62]. packings, and more recently with high-performance microparticulate supports [66, 67].