Separation and Analysis of Peptides and Proteins - Analytical

Anal. Chem. 1999, 71, 389R-423R

Separation and Analysis of Peptides and Proteins Cynthia K. Larive,*,† Susan M. Lunte,‡ Min Zhong,‡ Melissa D. Perkins,‡ George S. Wilson,†,‡ Giridharan Gokulrangan,† Todd Williams,§ Farhana Afroz,† Christian Scho 1 neich,‡ Tiffany S. Derrick,† ‡ ‡ C. Russell Middaugh, and Susan Bogdanowich-Knipp

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045 Review Contents Separation Methods Reversed-Phase Liquid Chromatography Salt-Promoted Adsorption Chromatography Normal-Phase Liquid Chromatography Ion-Exchange Chromatography Size-Exclusion Chromatography Affinity Chromatography Multidimensional Separations Ultrafiltration Two-Phase Systems for Protein Separation and Purification Protein Purification Capillary Electrophoresis Affinity Methods Mass Spectrometry Chemical Modification of Proteins Proteins in Cell Culture, Tissues, and Single Cells Nonenzymatic Posttranslational Modifications 3-Nitrotyrosine Protein Carbonyls Advanced Glycation End Products Other Oxidative Modifications Deamidation and Diketopiperazine Formation NMR Spectroscopy Comparison of NMR and X-ray Crystallography Structure Determination Membrane Proteins Pulsed-Field Gradient Methods Quantitative Analysis Analysis of Combinatorial Libraries HPLC NMR Vibrational Spectroscopy Infrared Spectroscopy Raman Spectroscopy Circular Dichroism Fluorescence Spectroscopy Ultraviolet/Visible Absorption Spectroscopy Light Scattering Calorimetry Literature Cited

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This review focuses on selected applications of the separation and analysis of peptides and proteins published during the period of 1997-1998. Specific topic areas covered include high-performance liquid chromatography (HPLC), ultrafiltration, capillary * Corresponding author: (phone) (785) 864-4269; (fax) (785) 864-5396; (email) [email protected]. † Department of Chemistry. ‡ Department of Pharmaceutical Chemistry. § Mass Spectrometry Laboratory. 10.1021/a1990013o CCC: $18.00 Published on Web 05/19/1999

© 1999 American Chemical Society

electrophoresis (CE), affinity-based methods for protein isolation and separation, mass spectrometry (MS), detection of nonenzymatic posttranslational modifications, nuclear magnetic resonance spectroscopy (NMR), infrared (IR) and Raman spectroscopy, circular dichroism (CD), UV-visible absorption spectroscopy, dynamic light scattering, and calorimetry. The quantification and identification of peptides and proteins by chromatographic methods and MS have become fairly routine, as has the conformational analysis of peptides and small proteins in solution by CD, IR, and NMR. Therefore, these topics are not reviewed in detail here. In this review, we have attempted to highlight new technological developments or unique applications of analytical methods that impact the analysis of peptides and proteins. SEPARATION METHODS High-performance liquid chromatography continues to be the method of choice for protein and peptide analysis. Over the past two years, several reviews have been published concerning advances in HPLC including equipment and instrumentation (A1) and theory (A2). New packing materials useful for protein chromatography have recently been reviewed by Leonard (A3). Reversed-Phase Liquid Chromatography. Reversed-phase liquid chromatography is a popular choice for the separation of peptides and proteins. In general, most articles are focused on new column support materials used to improve the speed of protein and peptide separations. These include perfusion stationary phases, which contain very large pores and nonporous materials consisting of very small diameter particles, ∼2 µm. Porous stationary phases have been recently reviewed by Rodriges (A4). The use of nonporous sorbents for protein separations has been reviewed by Lee (A5). Both types of chromatographic media can be used for fast separations. However, they also have limitations. Perfusion chromatographic media is less stable and the small particle size nonporous stationary phase can lead to high back pressures. Rippel et al. characterized nine commercially available silicabased, wide-pore reversed-phase columns using an acetonitrile/ water mobile phase and low-molecular-weight aromatic test compounds (A6). Physical chemical differences in the columns were determined using stationary-phase spectral mapping analysis (A6). A novel silica-based polymer-bonded packing material was described by Wei et al. (A7). The packing consisted of a macroporous silica support that was bonded with hydroxyethyl methylacrylate and divinylbenzene. It was stable from pH 2 to 10 and exhibited good mechanical strength. High-resolution separations of protein mixtures were achieved in less than 15 min (A7). Analytical Chemistry, Vol. 71, No. 12, June 15, 1999 389R

The optimization of column and run parameters for ultrafast protein separations is currently an area of focus for many investigators. Protein separations of less than 30 s were achieved using 20 × 4.6 mm columns filled with nonporous reversed-phase packing material (A8). The fast separation of proteins on 0.8- and 1.1-µm nonporous silica-based stationary phases has also been reported using milder conditions than those traditionally used for biopolymer separations (A9). Banks and Gulcicek compared protein separations on reversed-phase columns of nonporous silica to those obtained with conventional C18 material with a 300-Å pore size (A10). They were able to separate a nine-component peptide mixture in less than 35 s and a complete peptide map in 3.5 min using a nonporous reversed-phase column. A reversed-phase perfusion-based chromatographic method for the separation of phosphopeptides from their nonphosphorylated forms has been described (A11). A polymer-based reversed-phase Poros resin was employed with a pH 11.5 mobile phase. Phosphorylated peptides, a 15 mer and a 30 mer, and their nonphosphorylated forms were separated using this stationary phase where conventional separation methods using columns with silica-based reversed-phase packing material and an acidic mobile phase were unsuccessful. Improvements in the selectivity of protein separations by modification of the mobile phase were investigated by Sereda et al. (A12). It was demonstrated that the addition of hydrophilic anionic ion-pairing agents, such as perchlorate ion, to low-pH mobile phases can decrease the hydrophilicity of cationic side chains on proteins. This can lead to an enhancement in resolution as well as a change in selectivity. These authors also found that a phosphoric acid/perchlorate/acetonitrile mobile phase exhibited differences in selectivity from the aqueous trifluoroacetic acid/ acetonitrile mobile phases traditionally used for protein and peptide separations (A12). Temperature programming and gradient elution in reversedphase chromatography with 180-µm-i.d. packed capillary columns were compared for the separation of a mixture of proteins. Temperature programming was shown to be a useful approach to fine-tune protein separations and, in some cases, increase resolution (A13). Studies continue to be reported that investigate the mechanism of interaction of proteins with reversed-phase liquid chromatography stationary phases. The interaction of peptides with an n-alkylsilica-based, reversed-phase chromatographic stationary phase was modeled using a simulation based on molecular dynamics (A14). Salt-Promoted Adsorption Chromatography. This category includes hydrophobic interaction chromatography, thiophilic adsorption chromatography, and electron donor-acceptor chromatography. The thermodynamics of retention in hydrophobic interaction chromatography has been reviewed by Vailaya and Horva´th (A15). In this review, an emphasis was placed on the effect of temperature on the separation. A new hydrophobic interaction chromatographic support for protein separations has been described (A16). It consists of a continuous bed modified with isopropyl groups and was prepared for both conventional and microbore columns. The support consisted of piperazine diacrylamide, methacrylamide, and isopropylacrylamide. A gradient of ammonium sulfate in phosphate 390R

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buffer was used to achieve a high-resolution separation of seven proteins. Kanazawa et al. described a temperature-responsive chromatographic system for the analysis of peptides and proteins using an N-isopropylacrylamide polymer-modified column (A17). Aminopropyl silica was grafted to a temperature-responsive polymer, N-isopropylacrylamide, and its copolymer, butyl methacrylate, using the reaction of activated ester-amine coupling. The silica was then packed into a standard stainless steel column. A gradient elution-like effect was achieved using aqueous NaCl as the mobile phase. Higher temperatures led to an increase in retention time. The effect of the incorporation of hydrophobic sites in N-isopropylacrylamide polymer-modified columns was also investigated (A18). The hydrophobic sites were added to improve the selectivity and retention of the polymer-modified columns. The authors demonstrated reversed-phase-like separations by varying the temperature and mobile-phase composition with either water or aqueous sodium chloride (A18). N-Isopropylacrylamide and N-hydroxyethylacrylamide (70:30) were used to chemically coat wide-pore glass (mean pore diameter 2000 Å, particle size 0.16-0.31 mm). This material was then packed into 9 × 1 cm glass columns and used for the separation of proteins. The effect of temperature upon the separation was studied (A19). The characterization of the hydrodynamic and chromatographic properties of hydrophobic interaction chromatography columns is another area of study. In one study, macroporous poly(acrylamide-co-butyl methacrylate-co-N,N′-methylenebisacrylamide) monoliths containing up to 15% butyl methacrylate units were investigated. The separation of five proteins within 3 min was demonstrated. It was found that the hydrophobicity of the interacting surface can be controlled by the percentage of butyl methacrylate added (A20). In another study, a two-domain model was used to examine the retention behavior of proteins on hydrophobic interaction chromatography columns. The retention behavior was investigated as a function of mobile-phase ionic strength and column hydrophobicity (A21). Ruaan et al. discussed the potential for methacrylic block copolymers to be used as displacers or spacers in hydrophobic interaction chromatography (A22). In this displacement chromatographic mode, the substances of interest in the separation would be in direct competition with the copolymers for the adsorption site on the column. Trypsin and R-chymotrypsin were separated on an octyl-Sepharose hydrophobic interaction column. According to the authors, this is only the second report of an application of protein displacement in hydrophobic interaction (the first was Antia, F. D.; Fellegvari, I.; Horva´th, C. Ind. Eng. Chem. Res. 1995, 34, 2796-2804) (A22). De Frutos et al. studied the effect of chromatographic parameters on the peak shape of β-lactoglobulins A and B eluted in a hydrophobic interaction chromatography system. The peak shape changes were correlated with structural changes of the β-lactoglobulins (A23). Berna et al. performed the first study to directly compare the protein adsorption specificity of various agarose gels. Experiments were carried out by two-dimensional gel electrophoresis using human serum and aqueous solutions of varied concentrations of sodium sulfate. The differences in adsorbance due to hydrophobic interactions and/or electron donor-acceptor interactions are

discussed (A24). In a separate report, Gagnon et al. described an adaptation of hydrophobic interaction chromatography for the estimation of optimum protein solubility (A25). Normal-Phase Liquid Chromatography. Hydrophilic peptides are often not retained well on standard reversed-phase liquid chromatography column packing material. For this reason, normalphase liquid chromatography has been explored as a separation method for these peptides. The hydrophilicity retention coefficients of 121 peptides for normal-phase liquid chromatography were determined using a TSK Amide-80 column with carbamoyl groups bonded to a silica gel matrix (A26). A gradient separation consisting of increasing water in acetonitrile containing 0.1% trifluoroacetic acid was employed. The resulting coefficients can be used to predict retention times of peptides of a known sequence (A26). A normal-phase liquid chromatography method using TSK gel Amide-80, carbamoyl groups bonded to a silica gel matrix, and water/acetonitrile/trifluoroacetic acid mobile phase was established for hydrophilic peptides. Peptides were separated with good reproducibility and repeatability. As expected, the selectivity was shown to be very different from reversed-phase liquid chromatography (A27). Ion-Exchange Chromatography. Ion-exchange chromatography (IEC) is the most commonly practiced chromatography method for protein purification due to its ease of use and scaleup capabilities. In ion-exchange separations, the distribution and net charge on a protein’s surface determines the interaction of the protein with the charged groups on the surface of the packing materials. Three major groups of materials are used in the construction of ion exchangers: polystyrene, cellulose, and polymers of acrylamide and dextran. The most common functional groups for anion exchange are quaternary amines, diethylaminoethyl (DEAE), and polyethylenimine (PEI). For cation exchange, the most common groups are sulfpropyl (S or SP) and carboxyl (C or CM). A recent comprehensive review of the principles and applications of IEC to biological macromolecules and proteins has been published (A28). There are a large number of commercially available ionexchange adsorbents. Most possess the same active functional groups however; the method of attachment differs from column to column. To assist scientists in the development of ion-exchange separation protocols, Levison et al. compared the physical and functional performance of 70 different anion- and cation-exchange media including those based on polystyrene, cellulose, and polymers of acrylamide and dextran (A29). They found significant performance differences among the different ion-exchange media. This study is useful in terms of recommending particular ionexchange media for a given separation. The use of strong-cationexchange resins for the analysis and purification of peptides was recently reviewed by Crimmins (A30). This author achieved considerable success in the analysis and purification of well over 500 distinct synthetic and proteolytically derived peptide fragments using a sulfoethyl aspartamide SCX-HPLC. Several new packing materials have been explored in efforts to improve the performance over current ion-exchange materials. A poly(vinyl alcohol) (PVA)-coated particulate perfluoropolymer (FEP) support functionalized with ion-exchange groups was investigated for IEC of proteins (A31). It was demonstrated that

the PVA-FEP-based IEC could resolve proteins with only small differences in their isoelectric points (pI). The resolution of these proteins can be attributed to the minimization of mixed interactions in the synthesized ion-exchange materials. Zirconia-based polymeric ion-exchange stationary phases have also been reported for protein analysis. In one case, ethylenediamine-N,N′-tetramethylphosphonic acid (EDTPA), a phosphonate analogue of EDTA, was used as a surface modifier for zirconia (A32). The study showed that EDTPA-modified zirconia (PEZ) was a viable support for chromatography of highly basic proteins and for modulating the sites responsible for the high affinity of zirconia toward certain classes of anions. In another study, a new type of weak cation-exchange stationary phase was produced through immobilization of polyethyleneimine onto zirconia and functionalization with carboxylate groups. The resulting stationary phases are weak cation exchangers with a slight hydrophobic property. They provide unique selectivity for protein separations (A33). Lee et al. used zeolite X as a new stationary phase for IEC for the purification of immunoglobulin G (IgG) (A34). Zeolite X is an aluminosilicate crystalline material modified with calcium phosphate. These authors have demonstrated that zeolite X and dealuminated zeolite X are promising new packing materials for the purification of IgG from biological materials. The major problem with gradient elution IEC in the large-scale separation of protein products is that the scale-up and optimization process is usually determined by trial and error. This is timeconsuming and expensive. Theoretical models and computer programs are being developed to aid in the method development for protein separations using IEC. These models and programs have proven to be quite useful in the process optimization of protein purification using gradient elution IEC (A35-A38). The most important features to be determined in the separation of proteins are the separation resolution and recovery. The effects of separation conditions such as salt concentration in gradient elution (A39), pH, elution volume, and gradient profile (A40) on protein aggregation and resolution have been investigated. Protein adsorption onto solid surfaces has been characterized by Goheen and Hilsenbeck (A41). This information is useful in improving the understanding of both the chemistry and dynamics of protein adsorption in IEC and for providing structural information about the proteins themselves. Hydrolyzed carrageenan (A42), protected amino acids, antibiotics, dendritic polymers, and sucrose octasulfate (A43) have been reported as effective displacers for protein purification in IEC. It was demonstrated that the use of displacers in IEC results in high yields and production rated protein products. Gagnon et al. observed that the addition of polyethylene glycol (PEG) to the mobile phase of IEC significantly alters the retention behavior of proteins (A44). Because of its independence from the native selectivity of the ion exchanger, the addition of this polymer to the mobile phase created unique compound selectivity. However, the secondary effect of increasing viscosity by the addition of PEG has severely limited the preparative potential of its use. Size-Exclusion Chromatography. Size exclusion continues to be a very important protein separation tool. Size-exclusion chromatography and related separation techniques such as temperature-rising elution fractionation, field-flow fractionation, and enthalpic or interactive modes of high-performance liquid chroAnalytical Chemistry, Vol. 71, No. 12, June 15, 1999

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matography have been reviewed by Barth et al. (A45). The sizeexclusion high-performance separation of polypeptides of molecular weight less than 5000 has also been recently reviewed (A46). Ideally, size-exclusion chromatography should be performed such that the separation is based solely on size and there is no interaction of the sample with the packing material of the column. However, this is not always the case. Over the past two years, improvements in size-exclusion separations have been made by identifying mobile phases and mobile-phase additives, columnpacking materials, or modifications to packing materials that decrease these interactions. More fundamental studies have also been performed to better understand the nature of the interactions. Ricker and Sandoval reported techniques for optimization of size-exclusion chromatographic methods. Method parameters such as sample volume, flow rate, column length, and use of mobile-phase conditions that reduce nonideal interactions are considered (A47). Corradini studied the effect of electrostatic and hydrophobic interaction on the chromatographic behavior of biopolymers in chemically bonded silica-based HPLC columns designed for size-exclusion or anion-exchange chromatography. Aqueous buffered mobile phases containing neutral salts were used. The dominant interaction in the separation was found to be dependent on the concentration and nature of the salt in the mobile phase (A48). The ionic effects in size-exclusion chromatography and ultrafiltration were studied (A49). Correlations were made between the effective protein size as a function of ionic strength and the elution time of charged proteins separated by size-exclusion chromatography or neutral dextrans. A reduction in electrostatic shielding was demonstrated with increasing protein size and decreasing ionic strength. The importance of evaluating both steric and electrostatic interactions when determining protein retention times was shown. Theoretical models for the partitioning of charged solutes were used to quantify the results (A49). Modifying the silica support was found to reduce nonspecific adsorption of proteins to the silica-based column packing material during size-exclusion chromatographic runs. Porous silica beads were coated with dextrans of different molecular weights which had a calculated amount of DEAE groups and then grafted with a temperature-responsive polymer of poly(N-isopropylacrylamide) (PIPAAm). The results of these modifications were shown along with the response of the modified silica to temperature change (A50). Yu and He studied the effect of electrostatic interaction on the retention behavior of proteins in size-exclusion chromatography using nine proteins and varying the pH and/or ionic strength of the mobile phase (A51). The results indicated that for ideal SEC conditions the mobile-phase pH should be near the isoelectric point of the protein and the ionic strength should be high enough to weaken electrostatic interactions (0.1-0.3 M in this study). Ratto et al. demonstrated that the addition of 10% ethanol (v/v) to sodium chloride/phosphate aqueous mobile phases improved the performance of a silica-based size-exclusion chromatography column for the separation of poly(ethylene glycol) (PEG)-modified proteins. Also, their studies concluded that the interaction between the PEG-modified proteins and the silica stationary phase occurs through the PEG moiety (A52). 392R

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Magnetic resonance imaging (MRI) was used to visualize the chromatographic separation of proteins inside operating sizeexclusion and cation-exchange columns in order to characterize flow homogeneities and sample dispersion. This is the first application of MRI to visualize proteins in this manner (A53). In a different study, Wen et al. discuss a method to conveniently measure the extension coefficients of nonglycosylated proteins using refractive index and UV absorbance detectors and the application of these measurements for the study of protein interactions using size-exclusion chromatography (A54). Porath summarized his contributions to the field of separation science from gel filtration to adsorptive size exclusion (A55). Adsorptive size-exclusion chromatography was used to concentrate peptides and low-molecular-weight proteins from human plasma. This concentration effect was also demonstrated using ion-exchange and immobilized metal ion chromatography (A56). Affinity Chromatography. Affinity-based separations are popular for the separation of peptides and proteins due to their high specificity. This topic is treated in greater depth in the section on Affinity Methods. Numerous applications of affinity-based separations are reported in the literature as the initial step in protein purification. Some novel supports have recently been reported including the use of membrane supports to improve the speed of analysis (A57). Affinity chromatography followed by postcolumn immunochemical detection has been used for the separation and detection of recombinant methionyl granulocyte colony stimulating factor (GCSF) and GCSF modified with PEG. This method may prove to be useful for the measurement of recombinant proteins in body fluids (A58). Immunoaffinity approaches are also important in the field of proteomics where, for example, an immunoprecipitation step can be used to isolate specific protein-protein fusion products for further analysis by LC/MS (A59). Multidimensional Separations. The complexity of the molecular structures and the heterogeneous nature of proteins has necessitated the need for multidimensional separation techniques. There are two major areas of interest in the development of multidimensional separation techniques for proteins and peptides. The first area is the development of two-dimensional gel electrophoresis. Polyacrylamide is often used as the basic gel matrix (PAGE). The gel can be modified in terms of the degrees of cross-linking, addition of SDS, immobilization (covalent or noncovalent) of enzymes (affinity electrophoresis) or substrates (zymography), and pH gradient. This technique is often used for the characterization of proteins in terms of structural modifications, activities, pI values, and molecular weights. It has the advantages of high resolution, sensitivity, speed, reproducibility, and simplicity of operation. Feldstein et al. have developed a novel 2D denaturing PAGE system to completely separate a mixture of linear and circular RNAs containing 225-1132 nt (A60). Most 2D PAGE systems achieve resolution by increasing the concentration of either acrylamide or urea between the first and second dimension. In this report, the 2D-gel system relies on an increase in the degree of cross-linking to preferentially retard migration of circular molecules. Bruun et al. have presented a unique system that combines the use of small-scale hydrophobic interaction chromatography with 2D electrophoresis modified with pH gradients

(A61). The technique has been used for the isolation of serum amyloid A protein from small blood volumes (∼75 µL). Two-dimensional affinity electrophoresis has been used for the analysis of protein-protein interactions (A62, A63). In this case, a protein sample was first electrophoresed in a polyacrylamide gel, in which a bait protein of interest (calmodulin) had been immobilized. It was then separated by SDS-PAGE in the second dimension. If a sample protein interacts with the immobilized protein in the first dimension, it may migrate at a slower rate and a shift in mobility of the spot can be observed. Two-dimensional zymography (2-DZ) was used for analysis of proteolytic enzymes in human pure pancreatic juice (PPJ) (A64). 2-DZ was carried out by the combination of isoelectric focusing (IEF) in the first dimension with SDS-PAGE in the second dimension. The gels contain casein or gelatin as a substrate for enzymes in PPJ. This method was shown to be a successful tool for the clinical diagnosis of pancreatic cancer. Similar in concept to 2D gel electrophoresis, multidimensional chromatographic approaches are becoming more popular. Advantages include the ability to more accurately quantify the analyte and better compatibility with mass spectrometry. The two separation systems can be chosen to be orthogonal, leading to the greatest peak capacity. This chromatographic system makes it possible to directly couple the MS detector with the separation system as opposed to gel electrophoresis where analytes are bound to a matrix. However, the major technical hurdles are the integration of the two separation systems with the MS detector in terms of maintaining the resolution upon transfer to the second dimension and the compatibility of the mobile phases with the mass spectrometer. Opiteck et al. reported the first use of a comprehensive 2D LC/LC separation system coupled with MS detection for protein analysis (A65). This system used cation-exchange chromatography followed by reversed-phase chromatography. The two LC systems were coupled by an eight-port valve and two storage loops. The use of this LC/LC/MS system was demonstrated with the analysis of a mixture of standards and an Escherichia coli cell lysate. The major advantage of this technique is the on-line orthogonal separation by the 2D LC system. Subsequent reports have been published on 2D SEC/RPLC/MS for the analysis of tryptic digests of ovalbumin and serum albumin (A66) and for the separation of mixture of proteins resulting from the lysis of E. coli cells (A67). In a more recent report, Opiteck et al. described 2D SEC/RPLC/MS with microcolumn RPLC (A68). The use of microcolumn RPLC allows a lower flow rate that is more compatible with mass spectrometry detection. The increased peak concentration by the microcolumn RPLC also enabled higher detection sensitivity. Ultrafiltration. Protein fractionation by ultrafiltration (UF) has generated considerable commercial interest in the downstream separation of recombinant protein products and in blood plasma fractionation. Compared to chromatographic methods, membrane separation techniques offer advantages of lower cost and ease of translation to large-scale commercial production. However, the lack of selectivity and fouling of the membranes due to protein absorption during filtration has severely restricted the use of UF.

Novel UF membranes (A69) and the modification of UF membranes (A70, A71) have been reported. Torres et al. developed and characterized polyamide membranes for UF (A69). The operating conditions have been optimized and a potential application of these new membranes is the recovery of blood proteins such as hemoglobin or BSA in the dairy industry. Mathias et al. have developed a heterogeneous modification method for the attachment of reactive groups, suitable for covalent immobilization of active biomolecules on the surface of polysulfone ultrafilters without loss of membrane selectivity (A70). Covalent immobilization of a protein (bovine serum albumin, BSA), an enzyme (invertase), an antibody-enzyme conjugate (IgG-POD), and a peptide (“PCI”) as a specific antigen of a monoclonal antibody was demonstrated. High binding capacities were detected using these modified membranes. Stevens et al. studied the modification of UF membrane with gelatin protein to improve the flux and selectivity of the membrane in the presence of protein foulers (A71). Park et al. used a temperature-sensitive hydrogel for the separation of recombinant proteins (A72). Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration through gel swelling at 20 °C and collapsing at 35 °C. The attractive feature of this technique is that the hydrogel does not change the ionic environment or shear conditions of the medium during the operation. However, the separation efficiency and protein recovery is low due to the protein entrapment between gel particles and attachment of proteins to the gel surface. Wieboldt et al. explored the use of immunoaffinity UF in conjunction with ion spray HPLC/MS for screening of smallmolecule libraries (A73). The ultrafiltration process was used to isolate the affinity complexes from unbound molecules, and the filtrates containing the selectively captured compounds were analyzed by LC/MS. The direct on-line combination of UF with electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, was developed by van Breemen et al. (A74). The ligand-receptor complexes were purified by UF and then dissociated using methanol to elute the ligands for MS detection. This work has provided a simple and powerful new method for the screening of combinatorial libraries. Ghahramani et al. employed in vivo UF to determine protein binding of endogenous substances (A75). In vivo UF was found to be capable of accurately determining the protein binding of a drug. This method measures the total plasma concentration of drugs. It is useful in cases for which measurement of free drug is impossible because a sensitive assay is unavailable or when a drug is degraded rapidly in vitro. The poor selectivity of membranes has been regarded as one of the critical factors limiting the application of UF to protein fractionation. Gas sparging together with proper adjustment of solution conditions has proven to be able to dramatically improve the selectivity of a commercially available tubular PVDF membrane for the fractionation of the HAS/IgG mixture (A76). Another major factor limiting the use of this technique is membrane fouling. It is generally accepted that UF is not purely a size-based separation process and that other interactions such as membraneprotein and protein-protein interactions can significantly affect UF performance. Belfort et al. studied the intermolecular forces Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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between proteins and polymer films with relevance to filtration (A77). This study suggested that protein-protein and proteinpolymer interactions are equally important during UF and both the surface chemistry of the membrane and the solution conditions could be chosen to minimize fouling for specific protein solutions. Saksena and Zydney have developed a model to predict the influence of protein-protein interactions on bulk mass transfer during UF (A78). The model accurately predicted the large reduction in flux due to protein-protein interaction using BSA and IgG as an example. The effect of electrostatic interactions (A79, A80) and role of pH (A81) in the process of fractionation of proteins by UF have also been presented. In one study, Jaffrin et al. (A82) pointed out that the presence of ethanol in the solution enhances the fouling of the polysulfone hollow fiber membranes (30 kDa cutoff). Two-Phase Systems for Protein Separation and Purification. The use of hydrophobic partitioning of proteins in aqueous two-phase systems has been reviewed by Hachem et al. (A83). Protein surface tryptophans play an important role in the partitioning of proteins in aqueous two-phase systems. Such systems are used for the separation and purification of biological materials. The mechanism of the partitioning of proteins in aqueous twophase systems containing poly(ethylene glycol) and hydrophobically modified dextrans was studied. Particularly, the hydrophobic interactions between tryptophan residues and the hydrophobic groups on the modified dextrans were examined (A84). Studies of phase inversion were performed using aqueous two-phase systems for protein separation. Equilibrium binodal lines, tie lines, and phase inversion points were obtained using a poly(ethylene glycol) 400/phosphate system (A85). Protein Purification. Methods for protein purification of recombinant proteins were reviewed by Dyr and Suttnar (A86). In a separate review, Kaufman discussed the unique challenges and possible approaches for the purification of unstable proteins (A87). There are numerous applications of preparative chromatography cited in the literature. Instead of summarizing individual applications, we have chosen to emphasize new chromatographic supports, separation techniques, methodology, and modeling. Ultrastable zeolite Y was examined as a new matrix for protein purification. According to Beck, zeolites are tectosilicates consisting of corner-sharing AlO4 and SiO4 tetrahedra forming welldefined channels and cavities with aperture diameters ranging from 2.5 to 7.5 Å (A88). The zeolite used had a Si/Al ratio of >240. Protein-protein interactions played a role in layering the proteins on the surface of the ultrastable zeolite Y. Protein binding to the ultrastable zeolite was highest at or just below the pI of the protein; therefore proteins can be selectively bound to the zeolite creating a purification process for the remaining solution proteins (A89). Such methodology was demonstrated with the preparative gradient elution chromatography of chemotactic peptides using reversed-phase chromatography. The authors describe the theoretical as well as the experimental approach (A90). Strancar et al. presented a compact porous disk assembly for the fast separation of proteins on a preparative scale (A91). The assembly contains a compact porous disk as a separation unit. The separation can be operated in an anion-exchange, cationexchange, or hydrophobic interaction mode by changing the disks. 394R

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Three model proteins were separated within 1 min. This assembly can be used for on-line monitoring of production and downstream processing of biopolymers where fast analysis is highly desirable. A novel model for the blending of aqueous salt buffers for preparative chromatography was developed based on the simple continuously stirred tank reactor model. Predicted values from the model compared well with experimental data obtained. This model is particularly beneficial for preparative hydrophobic interaction and ion-exchange chromatography (A92). Countercurrent chromatography has also been employed for the preparative isolation of proteins. In the past two years, two reviews on this technique have appeared by Ito and Ma (A93, A94). Capillary Electrophoresis. Several reviews have been published on capillary electrophoresis of proteins and peptides. In particular, volume 271 of Methods of Enzymology was dedicated to high-resolution separations of biological macromolecules and includes capillary-based methods for the analysis of recombinant proteins (A95), as well as capillary electrophoresis with mass spectrophotometric detection (A96). In addition, several reviews on the use of CE for peptide and/or protein analysis have been published (A97-A99). These include two reviews covering theoretical predictions of peptide mobility as a function of pH (A100, A101). Modified Capillaries. One of the major areas of research in the separation of proteins with capillary electrophoresis is the development of new modified capillaries and run buffer additives to reduce absorption of proteins to the capillary walls. Lin and Hartwik investigated permanently poly(diallyldimethylammonium chloride)-modified capillaries over the pH range of 2.2-5.5 (A102). The capillaries generated fast anodal electroosmotic flow and improved efficiencies for basic proteins including lysozyme, myoglobin, and cytochrome c. A new method for the generation of coated capillaries using surface-confined living radical polymerization has been described by Wirth’s group (A103). The resulting coatings were evaluated for the analysis of strongly basic proteins and were found to have better reproducibility than linear polyacrylamide. In addition, there were no problems with clogging of the capillaries. A procedure for the production of cross-linked polymer coatings for CE has been described which involves simultaneous coupling and cross-linking (A104). The capillaries were evaluated for acidic and basic protein separations. Polyacrylamide (PA), polyvinylpyrrolidone (PVP), and poly(ethylene oxide) (PEO) were successfully anchored onto capillaries treated with several different silanes including methylacryloxypropyltrimethoxysilane (MET), chlorodimethyloctylsilane (OCT), and trimethoxyallylsilane. Acidic proteins were analyzed using an acrylamide-based cationic polymer capillary (MET-PVP). A new approach to the separation of acidic proteins at neutral pH was described by Katayama et al. (A105). In this approach, an anionic polymer is fixed to the capillary wall and a cationic polymer is sandwiched between the anionic polymer and the uncoated fused silica capillary by noncovalent bonding. In another report, a stable cationic capillary coating with successive multiple ionic polymer layers for the analysis of basic proteins is described (A106). The resulting capillary is stable in 1 M NaOH and 0.1 M HCl.

Polyetheleneimine-coated capillaries have been employed for the separation of basic proteins. It was found to be the best support for the separation of protein charge ladders obtained by acetylation of lysozyme (A107). The use of permanently modified capillaries is particularly important when interfacing with mass spectrometry. Covalently coated capillaries with polyethyleneimine have been found useful for CE/MS of proteins (A108). Polybrene-coated capillaries have also been evaluated for use in CE/MS (A109, A110). It was found that rapid analysis of hemoglobin variants could be obtained with better resolution using this method than with aminopropylsilane-modified capillaries. Alternative materials to fused silica have also been explored for biomolecule separations. Hydrophilic poly(methyl methyacrylate) (PMMA) hollow fibers were evaluated for the separation of hemoglobin variants (A111). It is not possible to monitor proteins at 210 nm because of the carbonyl groups on the PMMA capillaries absorb strongly below 250 nm however; detection limits for proteins at 280 nm are in the same range as for fused silica capillaries. Run Buffer Additives. Several types of additives have been explored for protein and peptide separations. Phytic acid continues to be useful for the separation of peptides and proteins (A112, A113). Phytic acid exhibits a low UV absorbance and due to its low conductivity can be used at concentrations up to 10 mM. It exhibits good buffer capacity between pH 2 and 11 and produces a significant reduction in analyte absorption to the capillary walls. Isoelectric buffers have also been investigated for peptide separations. Peptide maps are usually obtained in phosphate buffer, (pH 2). However, aspartic acid can be employed as an alternative buffering agent (A114). Using aspartic acid, higher voltages can be employed, leading to shorter run times. Some adsorption of the protein to the capillary wall can still occur and was eliminated in this report through the addition of 0.5% hydroxyethyl cellulose (A114). Cifuentes et al. performed a comparative study of coatings and additives including potassium chloride, morpholine, cetyltriethylammonium bromide (CTAB), poly(vinyl alcohol), and polyethyleneimine with model basic proteins including lysozyme, trypsinogen, and chymotrypsinogen (A115). They found CTAB gave the best performance with up to 300 000 theoretical plates/m obtained. Basic proteins have also been separated using a low concentration of CTAB (below the cmc). The optimal separation can be achieved by using the countermigration mode for the separation of peptides and proteins (A116). The effect of pH on separation was also investigated. Mixtures of surfactants have also been investigated as additives for protein and peptide separations (A117). By manipulating the ratios of a mixture of cationic and anionic fluorosurfactants, electroosmotic flow can be manipulated making it possible to produce efficient separations of acidic and basic proteins in a single run at neutral pH. Zwitterionic surfactants have been investigated for protein separations. They can both suppress the electroosmotic flow (EOF) and prevent wall adsorption. N-alkyl-N,N-dimethylammonio-1-propanesulfonates were investigated for the separation of basic proteins (A118). The EOF was suppressed 50-90% by dodecyldimethyl(3-sulfopropyl)ammonium hydroxide, hexadecyldimethyl(3-sulfopropyl)ammonium hydroxide, and coco(amidopropyl)hydroxyldimethylsulfobetaine at the critical micelle

concentration. Plate counts of 325 000-600 000 plates per meter were obtained under these conditions (A119). Micellar electrokinetic chromatography (MEKC) has been employed for peptide separations. Strenge and Lagu (A120) recently published a review of MEKC for the analysis of pharmaceutically relevant proteins. Two separate groups (A121, A122) investigated the separation of enkephalin analogues by MEKC. In one report, both SDS and CTAB were evaluated for the separation of enkephalin peptide analogues. It was found that the separation was based on both hydrophobicity and electrostatic interactions (A121). Strategies for optimization of MEKC separations of enkephalin-related peptides using multivariate analysis is described by Westerlund and co-workers (A122). The separation of cyclic peptides and their degradation products has also been accomplished by MEKC using SDS. The formation of oligomers due to cross-linking through the sulfur bridge was investigated using a combination of SEC and MEKC (A123). Wan and Blomberg describe separations of D-L peptides using teicoplanin as a chiral selector (A124). The reagent was found to be more specific for the D-form of the peptides. A separation of 15 D-L peptide derivatives was achieved using this reagent (A124). Enantiomers of di- and tripeptides using vancomycin as a chiral selector have also been described as well as the effect of organic modifier and buffer type on separation (A125). The use of sulfobutyl ether-derivatized β-cyclodextrin as a chiral discrimination reagent for the separation of acetylated Asp-Phe dipeptide stereoisomers has also been described (A126). Isoelectric focusing in the capillary format is becoming more widely accepted. One area of current research is the interfacing of capillary isoelectric focusing (CIEF) with mass spectrometry. Rodriquez-Diaz et al. discuss strategies to improve the performance of CIEF including elimination of reagents without buffering capacity, the use of internal markers and optimization of the CIEF mobilization procedure, the use of pressure, and the ion mobilization method (A127). The removal of salts by voltage ramping for on-line desalting of human blood and CSF samples prior to the CIEF separation of hemoglobin variants was described by Clarke et al. (A128). Future separation modes for proteins and peptides include electrochromatography and microfabricated analysis systems. Reports of the use of electrochromatography for the separation of proteins and peptides have appeared. These include the separation of peptides generated from tryptic digests of bovine β-lactoglobulin, human hemoglobin, and chicken ovalbumin using pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer. It was found that CEC gave faster more efficient separations than HPLC (A129, A130). Open tubular capillary electrochromatography has also been described for use with mass spectrometry for ultrafast peptide analysis (A131). Last, the development of macroporous polyacrylamide/poly(ethylene glycol) matrixes as stationary phases in CEC for the separation of peptides and carbohydrates has recently been reported by Palm and Novotny (A132). Microchip-based separation systems will play an important role in future peptide and protein separations, especially those needed in the field of proteomics or in high-throughput screening. Hoffman et al. reported isoelectric focusing of proteins on a glass chip (A133). Focusing of Cy5-labeled peptides was accomplished Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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in less than 30 s with plate heights of 0.4 µm. The total analysis time was 5 min with a peak capacity of 30-40. Duffy et al. described a method for rapid prototyping of microfluidic systems in poly(dimethylsiloxane) (PDMS) (A134). The resulting system was demonstrated for the separation of charge ladders of positively and negatively charged proteins, and the results were comparable to those obtained with fused silica capillaries. Sample Preparation Issues. Sample preparation is a very crucial issue for the analysis of proteins and peptides, especially if they are to be analyzed by capillary electrophoresis. Tomilinson et al. reviewed preconcentration and microreaction technology online with capillary electrophoresis including solid-phase extraction, membrane preconcentration, and microreactors (A135). The use of a C8 or C18 membrane has become popular for preconcentration and desalting of analyte prior to CE/MS (mPcCE/MS/MS). Using mPc, sample loading volumes 10000-fold greater than conventional CE can be used (A136). Membrane preconcentration has been used for the detection of histocompatibility complex class I peptides obtained from EG-& cells (A137). In another application of membrane concentration in combination with tandem mass spectrometry, peptide sequencing was accomplished at the sub-100 fmol level using a 100-µL sample that was loaded in less than 5 min (A138). Figeys et al. described solid-phase extraction using a Teflon sleeve packed with 5-µm C18 material for use in CE/MS (A139). Nine model peptides were investigated using solid-phase extraction capillary electrophoresis at low pH, with a C8 or C18 cartridge in line with an uncoated fused silica capillary. Peptides were released by introduction of an organic solvent. The solid-phase extraction device had a volume 20 times greater than the capillary volume (A140). The use of hollow fibers for preconcentration has become popular in CE. The Donnan effect can be exploited by immersing a hollow fiber filled with sample into a polymer solution and evaporation using a blow dryer or fan. Enrichment of proteins up to 3000-fold in one step has been accomplished via this technique (A141). Enrichment can also be accomplished by on-line electrophoretic concentration. In this case, samples are concentrated by placing a hollow fiber prior to the CE separation capillary. Proteins are concentrated inside the hollow fiber since they cannot pass through the membrane. Enrichment of model proteins by 1000fold has been reported (A142). Microdialysis sampling has been used in combination with CIEF and electrospray MS. The microdialysis membrane is used to remove the CIEF effluent from the ampholytes prior to MS analysis (A143, A144). Using this method mid-femtomole detection limits were obtained for carbonic anhydrase (A144). Smith’s group has used microdialysis extensively for the desalting of samples prior to introduction into the mass spectrometer. In one application, a microdialysis junction interface for mass spectrometry is described which avoids the need for makeup flow and the subsequent dilution in sensitivity. The system was demonstrated by postrun acidification of the CE effluent (A145). On-line microreactors have also been described for CE. Licklider and Kuhr described a trypsin-modified capillary microreactor with application of a mild vibration for enhanced rates of protein digestion prior to CE analysis (A146). 396R

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Affinity CE. Affinity CE (ACE) can be used to determine binding constants of proteins. This method has recently been reviewed by Heegaard et al. (A147) and by Rippel et al. (A148). In 1997, Busch et al. reviewed the principles and limitations of five methods for the determination of binding constants by ACE (A149). One recent application of affinity CE is the identification of epitopes on proteins. Epitopes can be determined using affinity capillary electrophoresis in conjunction with mass spectrometry. A tryptic map of a protein is generated by CE. The sample is then combined with the antibody and the CE analysis repeated. The location of the epitope can be determined by identifying which peptide peak disappears in the electropherogram. Only femtomole quantities of peptide are needed for the assay (A150). Capillary electrophoresis can also be used for the determination of proteins and peptides in biological fluids by a separationbased immunoassay. An on-line competitive immunoassay for insulin based on capillary electrophoresis with laser-induced fluorescence detection has been described by Tao and Kennedy (A151). The effective charge and molecular weight of proteins can also be estimated in solution by using capillary electrophoresis. Protein charge ladders are generated and can be used to estimate the effective charge (A152). AFFINITY METHODS The isolation and separation of proteins on the basis of molecular recognition has become an increasingly popular approach. The basis for recognition can be interactions typically found in nature such as antibody-antigen, receptor-antagonist, enzyme-substrate, or oligonucleotide-protein binding. It can also involve interactions with molecules such as dyes, metal complexes, RNA, or single-stranded DNA (ssDNA) modified to carry out specific functions. This approach is especially useful in situations where a high degree of specificity is required or where a particular protein, present at very low concentration, must be isolated from a cellular extract or other biological fluid. The use of combinatorial libraries has focused attention on biological recognition and on the detection of binding events that may be subsequently linked to biological function. One potentially useful approach to affinity chromatography involves the generation of a combinatorial library and forms the basis for a relatively new technique, systematic evolution of ligands by exponential enrichment (SELEX) (B1). This process, resulting in the preparation of aptamers (B2), a new class of biological recognition elements, is initiated with a random sequence ss-DNA or a RNA library of 1014-1015 sequences, of which ∼106 are likely to lead to useful interactions. The various sequences result in oligonucleotides with secondary motifs and tertiary structures that “fit” epitopes on proteins and therefore can bind with affinities comparable to those observed for antibodies (Kd e 10-8). The components of the library consist of a randomized sequence flanked by fixed sequences that facilitate amplification by PCR. After amplification, the resulting dsDNA is separated into ssDNA. For DNA SELEX, the amplified ssDNA library is then screened for binding against the target molecule and the binding DNA separated from the nonbinding species. The binding DNA is again amplified by PCR. This cycle is repeated 10-15 times to obtain enriched DNA with high specificity and affinity for the target molecule. Alternatively, the amplified ssDNA library can be

transcribed into RNA using T7 RNA polymerase if the original library contains a T7 promoter. The RNA thus produced is then screened against the target molecule. Binding RNA must then be converted back to DNA using a reverse transcriptase (RT) so that it can be amplified by PCR. A variety of methods are employed for separating the binding from the nonbinding aptamers, and it has recently been shown that magnetic beads can be used to capture the binding DNA which can amplified after dissociation from the beads (B3). Although the SELEX process seems complicated, preparation of suitable selective binding nucleotides can be accomplished in several weeks instead of the several months necessary to produce a monoclonal antibody. The biological recognition element can be either DNA or RNA; however, the latter are sensitive to degradation by ribonucleases. This problem has been addressed by Drolet (B4) and by Williams and co-workers (B5) through the introduction of forms of nucleic acids that are nuclease-resistant. Additionally nonbiological nucleic acids (biotinylated derivatives, for example) can be employed to facilitate subsequent binding to a substrate such as a column stationary phase. A wide variety of aptamers have been prepared to proteins (B6-B8) and to IFN-γ (B9), as well as a number of small molecules including individual amino acids (B10), hormones (B11), antibiotics (B12), dyes (B13), and therapeutic drugs (B14). Additional references and information may be found at the A. D. Ellington web site (http://speak.icmb.utexas.edu/cbss). It is also possible to carry out protein purification using DNA screened for specific binding of protein using band shift gels or footprinting. The DNA is covalently attached to a stationary phase using conventional chemistry such as CNBr-activated Sepharose. The isolation of protein from a cell extract, however, might consist of three steps: the first involves passing the cell extract over a heparin-agarose column, followed by a second column on which calf thymus DNA is immobilized, and finally a third column containing the DNA specific to a particular protein (B15). More strongly bound protein typically gives rise to the specific interactions, so proteins can be fractionated by an increasing salt gradient. Nucleosides can be immobilized on a support to form a γ-phosphoamide linkage and can then be used to purify enzymes such as ribonucleoside triphosphate reductase (B16). Antisense oligonucleotides have been used to purify human telomerase from nuclear extracts (B17). The use of peptide combinatorial libraries to optimize affinity chromatographic separations has not so far been widely applied. However, Buettner and co-workers (B18) have described methodology for screening bead-immobilized peptide libraries to identify suitable affinity agents. Antibodies continue to be widely employed in affinity chromatography. In some cases, the objective is the isolation of specific antibodies by immobilizing the antigen on a column, in others, the objective is to isolate and purify the antigen. The dissociation of the antibody-antigen complex to release the target molecule frequently presents problems. The conditions required for elution are often denaturing and can result in irreversible loss of biological activity of either the target molecule or the affinity stationary phase. Several workers have suggested buffers that offer the possibility of performing purification with good efficiency but with minimal loss of biological activity (B19, B20). A new stationaryphase material has proven extremely useful for affinity purifications

particle-loaded membranes (PLM) (B21). The membrane is ∼0.5 mm thick and contains 8-µm poly(styrene-divinylbenzene) (40% cross-linked) particles or 10-µm silica particles. Antibodies can be covalently attached to the particles and this gives rise to very efficient interactions between the mobile phase and the particle surfaces. For small molecules, recoveries of better than 90% are achievable in a mobile phase flowing at 70 mL/min. Comparable performance should be possible for proteins. It is well-known that glycoproteins can be purified using lectin affinity chromatography. Wheat germ aglutinin or concanavalin A are usually selected, but they have rather broad specificity, and therefore, the potential selectivity achievable with lectins cannot be realized. Since lectins are expensive and not very robust, Wiener and van Hoek devised a system for prescreening the target molecule against a series of fluorescent lectins in order that the optimal lectin can be chosen without having to prepare a series of columns (B22). Although proteins and nucleic acids can be very effective and powerful means to isolate and purify proteins, they are, in general, not practical for large-scale purification. They are not sufficiently robust and would be prohibitively expensive. One technique that continues to be very popular is immobilized metal affinity chromatography (IMAC), first reported by Porath in 1975 (B23). The most widely used support materials employ iminodiacetic acid or nitrilotriacetic acid (NTA) to which Ni(II) is typically chelated. This approach is especially appropriate to proteins produced by recombinant technology and to which a series of histidine residues (typically six) are sequentially genetically appended (B24). This has enabled the recovery of recombinant protein from inclusion bodies, which are formed as a consequence of the insolubility especially of membrane-bound species (B25). Attachment of NTA to magnetic beads provides a convenient means for carrying out rapid, small-scale purification of proteins (B26). IMAC has also been applied to the separation of ssDNA (B27). A tag consisting of six successive 6-histaminylpurine is added to the 5′-end of the DNA strand, thus permitting its recovery using Ni(II) IMAC after PCR. Attempts have also been made to combine IMAC with molecular imprinting (B28). Examples of applications to proteins based on “shape” recognition are, however, rather limited (B29). Immobilized dyes continue to be employed as affinity adsorbents. However, if the mobile phase is a cell or fermentation extract, it is likely to contain particulate matter, which would quickly clog the column. To deal with this problem a fluidized bed is employed. Such a procedure involves passing the mobile phase through a column in the direction opposite to gravity with the result that the “stationary-phase” particles become suspended. If the density of the particles is not very different from that of the mobile phase, then only low flow rates can be used. Pharmacia Bioprocess Technology AB has produced particles with a quartz core covered with cross-linked Sepharose (Streamline). The core increases the density and makes the particles much more suitable to fluidized-bed applications. Matiasson and co-workers attached Cibacron Blue 3GA to the particles (B30). Additionally, the surface was modified (polymer shielding) with poly(vinylpyrrolidone), which had previously been shown to minimize nonspecific interactions. The use of the fluidized bed and the lowered nonspecific interactions was tested in the purification of lactate Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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dehydrogenase from a porcine muscle crude extract. Significantly higher recoveries of active enzyme were the result. Finally, it is possible to take advantage of covalent affinity chromatography resulting from the formation of disulfide bonds between the stationary phase and a target molecule in the mobile phase. This approach was used to isolate and determine Cd metallothionein in human breast milk (B31). MASS SPECTROMETRY Mass spectrometry is a crucial tool in the analysis of peptides and proteins. In the companion Analytical Chemistry series of fundamental reviews, the authors of the mass spectrometry review provide an extensive presentation of the applications of MS for the analysis of peptides and proteins including ongoing summaries of the analysis of the most common protein posttranslational modifications and a compendium of peptide and protein molecular weights observed by MS (C1, C2). These offerings should be consulted for instrumental and technique developments. Matrixassisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are the dominant ionization methods, and the coupling of ESI to separation techniques is now standard (C2). Two themes to be pursued in this section are the use of MS to obtain information on the functional aspects of proteins and their interactions in a biological environment. Chemical Modification of Proteins. Chemical modification of proteins has been utilized as a probe of protein secondary structure or active site characterization, and MS is the method of choice to analyze the products of such reactions. An inhibitor of monoamine oxidase B, N-cyclopropyl-R-methylbenzylamine, was found to modify Cys-365 in the bovine liver enzyme. Analysis was by HPLC/MS of lysine C digest (C3). Diethyl pyrocarbonate (DEP) was used to modify histidine residues, and the carbethoxylated derivatives in angiotensin II and insulin were characterized (C4). DEP was used for selective modifications of histidine, tyrosine, and lysine in recombinant human macrophage colonystimulating factor. A loss of activity was correlated with modification of His-9 and His-15 (C5). Lysine 207 was shown to be the cross-linking site between the 3′-end of E. coli initiator tRNA and methionyl-tRNA formyltransferase (C6). Capillary LC/ESI MS was used to identify a specific S-thiolation-susceptible cysteine residue in recombinant human Cu, Zn superoxide dismutase (C7). Lysine 464 in Ca-ATPase in skeletal sarcoplasmic reticulum membranes was selectively labeled with the fluorescent probe erythrosin isocyanate (C8). In vitro nitration of Tyr-164 and Tyr-166 of surfactant protein A was found to correlate with a decreased ability to aggregate with lipids (C9). A photoaffinity label was used to locate the substrate recognition region in mammalian protein farnesyltransferase (I) as Asp-110 to Arg-112 in the R-subunit (C10). Fluorescent tags have been incorporated in porcine neuropeptide Y, and the analogues are recognized by receptors (C11). Acetaldehyde was found to produce stable imidazolidinone peptide and protein adducts which may serve as biomarkers for alcohol exposure (C12). Secondary structure elucidation by particle beam LC-FT-IR and LC/MS for β-lactoglobulins A and B has been reported (C13). The kinetics of cytochrome c folding has been examined by monitoring pulsed H/D exchange by ESI MS (C14). MS analysis revealed that correctly folded, active 398R

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crystallizable human interferon-R-8 was a mixture of “native” and N-acylated protein (C15). MALDI-MS has been used to identify contact sites between ribosomal proteins and rRNA in E. coli in cross-linking studies (C16, C17). Trifluoromethionine (TFM) was found to be resistant to CNBr/formic acid cleavage when TFM was incorporated in recombinant lysozyme. Formylation products were observed on some serine, threonine, and C-terminal homoserine residues (C18). The reactions of the group-specific reagents, phenylglyoxal, trinitrobenzenesulfonic acid, tetranitromethane, and diethyl pyrocarbonate was correlated with enzyme inactivation and active sites determined by MS analysis of modified proteins in the shikimate pathway (C19). Cyanylation of Cys yields a modified amino acid amenable to specific cleavage. Optimization of this cleavage chemistry has been studied and applied to a variety of proteins (C20). An alternative disulfide bond assignment strategy has been demonstrated using the cyanylated Cys cleavage reactions (C21). MALDI-MS has also been used to determine disulfide bond linkages and structures in human epidermal growth factor receptor (C22), Plasmodium apical membrane antigen-1 (C23), and Fel d1 (C24) cat allergen. Proteins in Cell Culture, Tissues, and Single Cells. The sensitivity of instruments utilizing MALDI and ESI makes them useful tools for monitoring processes in cell culture. In an example of subfemtomole sensitivity, an antigen peptide was detected from melanoma tumor cells by MS and MS/MS. The isolation/ separation technique used was membrane preconcentration for transient isotachophoresis capillary electrophoresis prior to ESI in an ion trap instrument (C25). While analysis of overexpressed proteins is now common, MS has been proposed to monitor expression by sampling whole bacteria. MALDI provided advantages over gel electrophoresis when compared for efficiency, resolution, mass accuracy, and information content (C26). SDS/ gel CE and MALDI-MS were used to monitor changes in culture broth, including an off-line strategy for coupling of CZE to MS (C27). Proteins excreted into media were used to monitor the product yield and culture viability of several mammalian cell lines by MALDI. Sample preparation strategies included detergents that increased detectable masses to 75 kDa (C28). Some of the soluble peptides derived from amyloid (β) protein were detected in culture media from mouse neuroblastoma cells transfected with human amyloid precursor protein. Identification and quantification were demonstrated (C29). Protein expression patterns in cardiac myocytes were used to monitor a culture model of hypertrophy. Silver staining of low-SDS-content 2D gels improved peptide recovery from in-gel digest for analysis by MALDI or HPLC/MS (C30). Cytokine-regulated proteins in cervical carcinoma cells were identified from samples prepared in 2D gels in which the first-dimension isoelectric focusing was obtained in an immobilized pH gradient followed by the traditional sizing in the second dimension. Protein identification of the imidazole-zinc stained spots was by MALDI-TOF analysis of gel digests. Regulated proteins found were triosephosphate isomerase, subunit C3 of the proteosome, and the GTP-binding protein Ran (C31). A related application area is the analysis of tissues. MALDITOF has been used as an analytical method for imaging biological samples in two dimensions. Examples are the detection of insulin in sections of pancreas, hormones in the pituitary, and small

proteins on the membrane of mucosa cells (C32). Peptides have been detected in isolated cells and tissue from the nervous system of marine mollusks. The 2,5-dihydroxbenzoic acid matrix required for MALDI also served to stabilize cell membranes, deactivate endogenous proteases, and overcome salt contamination (C33). Human and salmon calcitonan have been used as reporter peptides to study metabolism in bovine nasal mucosa (C34). Two precursors of the hyperglycemic hormone from neurosecretory cells were detected both by MALDI and by immunodetection with ELISA in the same samples (C35). MS-based strategies have been applied to peptide analysis of single cells from the nervous system of the pond snail Lymnae stagnalis. Single neurons from the brain were analyzed for peptide content using microcolumn LC. The column effluent was deposited onto the target stage of a MALDI-TOF instrument (C36). Peptide masses detected in a piece of penis nerve correspond to cardioactive peptides previously identified in this species, and the sequences were confirmed with MS/MS directly from a single neuron. These peptides were found elsewhere in the nervous system and were found to be active in a bioassay (C37). Cardioactive peptides involved in the modulation of heartbeat were identified in neurons. These sequences were used to synthesize an oligonucleotide that was used to isolate the cDNA encoding the precursor to the cardioactive peptides from a brain-specific cDNA library (C38). NONENZYMATIC POSTTRANSLATIONAL MODIFICATIONS Protein degradation by nonenzymatic posttranslational modifications plays an important role in vivo, for example, during biological conditions of oxidative stress, as well as in vitro, for example, during the manufacturing, isolation, and storage of biotechnology products. We continue to see a large body of work concerning the characterization and method development for the exact quantification of such posttranslational modifications in order to obtain biomarkers for various pathologies or impurity profiles of protein formulations. Here we will focus mainly on the analysis of 3-nitrotyrosine, protein carbonyls, and advanced glycation endproducts (AGE). 3-Nitrotyrosine. On the basis of the finding that the incubation of tyrosine with peroxynitrite yields 3-nitrotyrosine (3-NT), the presence of 3-NT in tissue was taken as evidence that peroxynitrite was formed in vivo. However, more recent studies have characterized additional pathways of tyrosine nitration through ClNO2 (D1), myeloperoxidase/H2O2/NO2- (D2, D3), horseradish peroxidase/H2O2/NO2- (D3), and HOCl/NO2- (nitration of low-density lipoprotein (LDL)) (D4). Singh et al. (D5) demonstrated the nitration of another phenolic compound, γ-tocopherol, by superoxide dismutase (SOD)/H2O2/NO2-, and it may be concluded that such a system would potentially also nitrate tyrosine. The presence of 3-NT in tissue should, therefore, be taken as general evidence for the formation of reactive nitrogen species in vivo (D6). The analysis of 3-NT requires a critical comparison of the data to carefully executed controls. Western blot analysis of 3-NT-containing proteins must be performed before and after reduction of 3-NT to 3-aminotyrosine (3-AT) with dithionite (D7). Attomole levels of 3-AT can, in fact, be quantified by stable isotope dilution gas chromatography/mass spectrometry (D8). However, even the reduction of 3-NT to 3-AT failed to serve

as a control in the direct HPLC analysis of 3-NT in human postmortem brain tissue derived from patients with Parkinson’s disease, Huntington’s chorea, multiple system atrophy, and Alzheimer’s disease (D9). Further monitoring of the peak coeluting with authentic 3-NT by amperometric detection and mass spectrometry failed to confirm the identity of this peak as 3-NT. Sensitive methods for the electrochemical detection of 3-NT have been developed by Shigenaga et al. (D10) (after reduction to 3-AT), Althaus et al. (D11), and Hensley et al. (D12) (see also Erratum (D13)). If sample quantities permit, protein tyrosine nitration is best characterized by mass spectrometric analysis of a purified complete or proteolytically digested peptide or protein, such as performed for some model peptides (D14), angiotensin (D15), MnSOD (D16, D17), and neurofilament-L (D18). Protein nitration can be chemically quite selective such as demonstrated for two isoforms of the sarcoplasmic Ca-ATPase in Fischer 344 rat skeletal muscle where the SERCA2a but not the SERCA1 isoform shows nitration as a consequence of biological aging (D19, D20). Interestingly, when Leeuwenburgh et al. (D21) screened rat skeletal muscle homogenate for the age-dependent accumulation of 3-NT, they did not find significant differences between young and old female Long-Evans/Wistar rats. Thus, depending on the specificity for selected proteins and their abundance, molecular chemical changes such as 3-NT accumulation on such proteins may not be detectable through the analysis of whole tissue homogenates or extracts. In a Parkinson’s disease model, administering 1-methyl-4-phenylpyridinium (MPP+) to PC12 cells, Ara et al. reported a selective nitration of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis (D22). Complicating the analysis of protein-bound 3-NT in vivo may be a recently described “nitrotyrosine denitrase” activity which, despite the presence of protease inhibitors, removed the epitope recognized by the anti-nitrotyrosine antibody from some proteins but only inefficiently from free 3-NT (D23). By immunostaining, protein nitration has been documented as a consequence of focal ischemia (D24), in postischemic cerebral cortex (D25), in Alzheimer’s disease brain (D26, D27), in RcsX lymphoma of SJL mice (D28), on β-VDLD apoproteins in an experimental atherosclerosis model (D29), in plasma of premature infants developing bronchopulmonary dysplasia (D30), in the liver following toxic doses of acetaminophen (D31), and in human plasma (D32). 3-NT was found in the upper and lower spinal cord and the cerebral cortex of transgenic mice expressing the G93A mutation of Cu,ZnSOD (D33). Similarly, 3-NT levels were enhanced in transgenic mice expressing the G37R mutation of Cu,ZnSOD (D34). Both mutations are, among others, associated with familial amyotrophic lateral sclerosis (FALS). Significantly increased levels of 3-NT were also localized to the spinal cords of patients with sporadic cases of ALS (D35). Protein Carbonyls. The measurement of protein carbonyls is a sensitive albeit rather nonspecific assessment for the oxidative damage of proteins. Generally, protein carbonyls form through a direct conversion of an amino acid side chain into a carbonyl or through the covalent attachment of carbonyl-carrying molecules such as 4-hydroxynonenal. The detection of protein carbonyls is possible after derivatization with 2,4-dinitrophenylhydrazine (2,4DNPH) and either spectrophotometric analysis or staining with an anti-dinitrophenyl (DNP) antibody (D36). These methods Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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served to monitor higher oxidative stress in the substantia nigra (as compared to other brain regions) (D37) and in Parkinson’s disease brain (D38). We note that Focht et al. reported no significant changes in protein carbonyl content for nine brain regions as a result of biological aging of Fischer 344 rats (D39) whereas Aksenova et al. (D40) confirmed an age-dependent higher carbonyl formation in the brain of male and female brown Norway rats, affected by dietary restriction. The controversy over the extent of age-associated accumulation of protein carbonyls is the theme of a review by Goto and Nakamura (D41). Higher contents of carbonyls as compared to controls were detected in the motor cortex, parietal cortex, and cerebellum of FALS and spontaneous amyotrophic lateral sclerosis (SALS) patients (D42), in the parietal lobe of Alzheimer’s disease patients (D43) (see also review by Markesbery (D44)), in the parietal and temporal lobe of patients suffering dementia with Lewy bodies (DLB) (D45), in experimental acute pancreatitis (D46), and for the sarcoplasmic reticulum Ca-ATPase of low-frequency-stimulated rabbit muscle (D47). Mecocci et al. found an age-dependent increase of protein carbonyls in human skeletal muscle (D48). Specifically, a few proteins were identified as major targets for oxidative stress when E. coli were exposed to several types of oxidative stress (D49), and protein carbonyls have been detected in bovine aortic endothelial cells exposed to hydrogen peroxide or the xanthine/xanthine oxidase system (D50). Buss et al. developed an ELISA method for the detection of protein carbonyls based on 2,4-DNPH labeling followed by probing with a biotinconjugated primary anti-DNP antibody and detection with streptavidin-linked horseradish peroxidase (D51) (see also Erratum (D52)). Instead of labeling with 2,4-DNPH, protein carbonyls have also been labeled with digoxigenin-hydrazide and stained with an anti-digoxigenin antibody (D53). As an alternative method, protein carbonyls can be quantified via the alcohol dehydrogenasecatalyzed reduction by NADH (D54). Advanced Glycation End Products. The formation of advanced glycation end Products (AGE) represents an important covalent protein modification associated, for example, with aging and diabetes. In healthy human articular cartilage, pentosidine levels increase linearly after 20 years of age (D55). Similarly, human lens proteins experience increasing age-dependent modification by methylglyoxal to N--carboxymethyllysine (D56). Aging results in the formation of early glycation Amadori products such as furosine in rodent skin collagen (D57). Odetti et al. report that AGEs are significantly elevated in the cortex of fetal brain of Down’s syndrome subjects (D58), and Li and Dickson find high yields of AGEs in Alzheimer’s disease brain, colocalizing with apolipoprotein E (apoE) (D59). However, Tabaton et al. report that neither amyloid β protein nor apoE is glycated in vivo (D60) even though the nucleation-dependent aggregation of amyloid β protein in vitro can be accelerated through glycation (D61). In contrast, Seidl et al. conclude that AGEs are not more abundant in the frontal cortex of Down’s syndrome and Alzheimer’s disease patients as compared to controls (D62). Mitsuhashi et al. point out that the lack of suitable AGE standards could be a problem for the comparative quantitation of AGEs between research laboratories. They suggest utilizing AGE units against normal human serum (D63). Antibodies specific for pyrraline and pentosidine showed higher levels of glycation in Rosenthal fibers of 400R

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patients with Alexander’s disease (D64). IgG appears to be a predominant target for the accumulation of AGEs in plasma of an experimental diabetes model (D65), Lapolla et al. demonstrated increased levels of IgG glycation in plasma of diabetic vs control patients by MALDI mass spectrometry (D66). Serum proteins of diabetic individuals reacted more strongly with an antibody recognizing imidazolysine and argpyrimidine as compared to normal individuals (D67), and imidazolone is present at high levels in kidneys of rats with streptozotocin-induced diabetes (D68) and in diabetic patients (D69). Higher levels of N--carboxymethyllysine-protein adducts are present in peripheral nerves of human diabetics (D70). Wrobel et al. examined plasma from normal and diabetic individuals in a flow system connected on-line with a spectrophotometric and a fluorescence detector for the simultaneous monitoring of peptides and AGEs (D71). In vitro glycation of LDL leads to the modification of multiple sites including two protein domains adjacent to the apoB domain important for binding to the LDL receptor (LDLr), a fact that may be responsible for the decrease of LDL binding to the LDLr (D72). Separation and identification strategies for glycated proteins have been discussed by Deyl and Miksik (D73, D74). Pentosidine-containing peptides derived from a CNBr digest of modified collagen have been separated on a C4 macroreticular sorbent (D75). Mass spectrometry has been utilized to identify imidazolium cross-links formed by the reaction of Lys with glyoxal and methylglyoxal (D76), and methylglyoxal has been shown to be present at high levels, mostly bound to biological ligands, in CHO cells (D77). Methylglyoxal-dependent modifications are further present in atherosclerotic lesions of human aorta (D78). Liquid chromatography/electrospray tandem mass spectrometry served to detect and characterize lactolated peptides (D79) Electrospray and MALDI-TOF mass spectrometry methods for the direct analysis of glycated proteins including glycohemoglobin have been developed (D80-D83). Oya et al. identified 8-hydroxy-5-methyldihydrothiazolo(3,2-a)pyridinium-3-carboxylate (MRX) as a novel biomarker for hyperglycemia (D84). Gordon et al. characterized N8(4-oxo-5-dihydroimidazol-2-yl)-L-ornithine as a product from the in vitro reaction of ribose and collagen (D85). Threosidine, a 2,5dihydroxy-5,6,7,8-tetrahydro-1,7-naphthyridinium derivative, was identified as a novel class of Lys-Lys cross-links through reaction with L-threose (D86). Nakamura et al. (D87) characterized vesperlysines A, B, and C as major AGEs derived from the HCl hydrolysis of AGE-BSA. Cross-linking of proteins occurs predominantly when Lys -amino groups are glycated, supported by experimental results obtained with Lys2Thr and Lys163Thr mutants of γ-B-crystalline (D88). Slatter et al. characterized a dihydropyridine derivative formed through reaction between malondialdehyde and Lys using NMR (D89). Fu et al. demonstrated that the incubation of collagen with glucose under oxidizing conditions not only leads to the accumulation of AGEs but also to other amino acid oxidation products such as m-tyrosine, dityrosine, dopa, and valine and leucine hydroperoxides (D90). Other Oxidative Modifications. Garner et al. have identified that the oxidation of Met to methionine sulfoxide in apolipoproteins I and II is an early event accompanying lipid peroxidation of high-density lipoprotein (HDL) (D91, D92). Oxidation of Met to methionine sulfoxide as well as Tyr to dityrosine results in a significant increase of the hydrophobicity of liver proteins, as

analyzed by binding of 8-anilino-1-naphthalenesulfonic acid (D93). Fu et al. monitored cataract samples for oxidation products resulting from the reaction of hydroxyl radicals. They found two characteristic products, 3-hydroxyvaline and 5-hydroxyleucine (for the structural characterization of hydroxyl radical oxidation products of Leu, see ref D94), besides 3,4-dihydroxyphenylalanine (dopa), o- and m-tyrosine, and bityrosine (D95). The presence of protein-bound dopa in vivo may initiate further oxidative damage, e.g., of DNA (D96). Ad libitum fed mice showed increasing levels of o,o-dityrosine with age in cardiac and skeletal muscle but not in liver and brain, which was attenuated by caloric restriction, whereas o-tyrosine levels did not show an age-dependent increase in all these tissues (D97). In contrast, Wells-Knecht et al. detected increased levels of o-tyrosine in human collagen (D98). Another amino acid especially sensitive to metal-catalyzed oxidation is His. Retsky et al. demonstrated the oxidation of His in LDL to 2-oxoHis by ascorbate/Cu(II) (D99). In a more mechanistic study, the participation of hydroxyl radicals in the oxidation of His to 2-oxoHis was established for the metal-catalyzed oxidation of His in human growth hormone (D100). Deamidation and Diketopiperazine Formation. Various examples of the identification of deamidation sites in peptides and proteins have been presented such as for charge isomers of bovine myelin basic protein (D101), a recombinant hepatitis E vaccine candidate (D102), recombinant human parathyroid hormone (D103), recombinant human stem cell factor (D104), pramlintide (D105), mammalian cAMP-dependent protein kinase (D106), and insulin (D107). Diketopiperazine formation involves an intramolecular aminolysis of the first two N-terminal residues of a protein and mechanistic studies have been undertaken employing small model peptides (D108-D110). NMR SPECTROSCOPY NMR is one of the premiere analytical techniques for structure elucididation. The application of multidimensional NMR methods for the resonance assignment and structure determination of peptides and small proteins in solution has become routine and will not be discussed in detail in this review. Rather we have elected to focus on new developments in the measurement and analysis of protein structures. Applications of NMR for affinity measurements and for the analysis of combinatorial libraries are also presented. Comparison of NMR and X-ray Crystallography. Until recently, the three-dimensional structure of proteins and nucleic acids at atomic resolution had largely been determined using X-ray crystallography. The situation began to change in 1984 when the first 3D structure of a globular protein was solved using NMR (E1). Since then, NMR has become a routine method for the elucidation of macromolecular 3D structures as evidenced by the fact that 21-36% of all determined structures have been solved by NMR methods. NMR is well suited for smaller proteins (30 000) can be aligned. This is achieved using a method based on isopotential spin-dry ultracentrifugation (ISDU) (E35). This method is capable of preserving the biological integrity of the sample while giving good membrane alignment. It was demonstrated using three different families of proteins that range in molecular size from 39 to 100 kDa. The results of the alignment as assessed by static 31P NMR showed that all but one of these proteins could be oriented in their native membranes with sufficient alignment. The quality of the alignment was determined to be a “mosaic spread” (E35). The mosaic spread is used to describe the line broadening associated with orientational defects resulting from membranes that are not highly ordered. To correct for the mosaic spread, Glaubitz and Watts used a method called magic angle-oriented sample spinning (MAOSS) (E36) in which uniformly aligned biomembrane samples are combined with MAS at low speeds (