Preface All areas of the biological sciences have become increasingly molecular in the past decade, and this has led to ever greater demands on analytical methodology Revolutionary changes in quantitative and structure analysis have resulted, with changes continuing to this day Nowhere has this been seen to a greater extent than in the advances in macromolecular structure elucidation This advancement toward the exact chemical structure of macromolecules has been essential in our understanding of biological processes This trend has fueled demands for increased ability to handle wmishingly small quantities of material such as from tissue extracts or single cells Methods with a high degree of automation and throughput are also being developed In the past, the analysis of macromolecules in biological fluids relied on methods that used specific probes to detect small regions of the molecule, often in only partially purified samples For example, proteins were labeled with radioactivity by in vivo incorporation Another approach has been the detection of a sample separated in a gel electrophoresis by means of blotting with an antibody or with a tagged oligonucleotide probe Such procedures have the advantages of sensitivity and specificity The disadvantages of such approaches, however, are many, and range from handling problems of radioactivity, as well as the inability to perform a variety of in vivo experiments, to the invisibility of residues out of the contact domain of the tagged region, e.g., epitope regions in antibody-based recognition reactions Beyond basic biological research, the advent of biotechnology has also created a need for a higher level of detail in the analysis of macromolecules, which has resulted in protocols that can detect the transformation of a single functional group in a protein of 50,000-100,000 daltons or the presence of a single or modified base change in an oligonucleotide of several hundred or several thousand residues The discovery of a variety of posttranslational modifications in proteins has further increased the demand for a high degree of specificity in structure analysis With the arrival of the human genome and other sequencing initiatives, the requirement for a much more rapid method for D N A sequencing has stimulated the need for methods with a high degree of throughput and low degree of error The bioanalytical chemist has responded to these challenges in biological measurements with the introduction of new, high resolution separation and detection methods that allow for the rapid analysis and characterization of macromolecules Also, methods that can determine small differences in xii PREFACE many thousands of atoms have been developed The separation techniques include affinity chromatography, reversed phase liquid chromatography (LC), and capillary electrophoresis We include mass spectrometry as a high resolution separation method, both given the fact that the method is fundamentally a procedure for separating gaseous ions and because separation-mass spectrometry (LC/MS, CE/MS) is an integral part of modern bioanalysis of macromolecules The characterization of complex biopolymers typically involves cleavage of the macromolecule with specific reagents, such as proteases, restriction enzymes, or chemical cleavage substances The resulting mixture of fragments is then separated to produce a map (e.g., peptide map) that can be related to the original macromolecule from knowledge of the specificity of the reagent used for the cleavage Such fingerprinting approaches reduce the characterization problem from a single complex substance to a number of smaller and thus simpler units that can be more easily analyzed once separation has been achieved Recent advances in mass spectrometry have been invaluable in determining the structure of these smaller units In addition, differences in the macromolecule relative to a reference molecule can be related to an observable difference in the map The results of mass spectrometric measurements are frequently complemented by more traditional approaches, e.g., N-terminal sequencing of proteins or the Sanger method for the sequencing of oligonucleotides Furthermore, a recent trend is to follow kinetically the enzymatic degradation of a macromolecule (e.g., carboxypeptidase) By measuring the molecular weight differences of the degraded molecule as a function of time using mass spectrometry [e.g., matrix-assisted laser desorption ionization-time of flight ( M A L D I - T O F ) ] , individual residues that have been cleaved (e.g., amino acids) can be determined As well as producing detailed chemical information on the macromolecule, many of these methods also have the advantage of a high degree of mass sensitivity since new instrumentation, such as M A L D I - T O F or capillary electrophoresis with laser-based fluorescence detection, can handle vanishingly small amounts of material The low femtomole to attomole sensitivity achieved with many of these systems permits detection more sensitive than that achieved with tritium or 14C isotopes and often equals that achieved with the use of 32p or 125I radioactivity A trend in mass spectrometry has been the extension of the technology to ever greater mass ranges so that now proteins of molecular weights greater than 200,000 and oligonucleotides of more than 100 residues can be transferred into the gas phase and then measured in a mass analyzer The purpose of Volumes 270 and 271 of Methods in Enzymology is to provide in one source an overview of the exciting recent advances in the PREFACE xiii analytical sciences that are of importance in contemporary biology While core laboratories have greatly expanded the access of many scientists to expensive and sophisticated instruments, a decided trend is the introduction of less expensive, dedicated systems that are installed on a widespread basis, especially as individual workstations The advancement of technology and chemistry has been such that measurements unheard of a few years ago are now routine, e.g., carbohydrate sequencing of glycoproteins Such developments require scientists working in biological fields to have a greater understanding and utilization of analytical methodology The chapters provide an update in recent advances of modern analytical methods that allow the practitioner to extract maximum information from an analysis Where possible, the chapters also have a practical focus and concentrate on methodological details which are key to a particular method The contributions appear in two volumes: Volume 270, High Resolution Separation of Biological Macromolecules, Part A: Fundamentals and Volume 271, High Resolution Separation of Biological Macromolecules, Part B: Applications Each volume is subdivided into three main areas: liquid chromatography, slab gel and capillary electrophoresis, and mass spectrometry One important emphasis has been the integration of methods, in particular LC/MS and CE/MS In many methods, chemical operations are integrated at the front end of the separation and may also be significant in detection Often in an analysis, a battery of methods are combined to develop a complete picture of the system and to cross-validate the information The focus of the LC section is on updating the most significant new approaches to biomolecular analysis LC has been covered in recent volumes of this series, therefore these volumes concentrate on relevant applications that allow for automation, greater speed of analysis, or higher separation efficiency In the electrophoresis section, recent work with slab gels which focuses on high resolution analysis is covered Many applications are being converted from the slab gel into a column format to combine the advantages of electrophoresis with those of chromatography The field of capillary electrophoresis, which is a recent, significant high resolution method for biopolymers, is fully covered The third section contains important methods for the ionization of macromolecules into the gas phase as well as new methods for mass measurements which are currently in use or have great future potential The integrated or hybrid systems are demonstrated with important applications We welcome readers from the biological sciences and feel confident that they will find these volumes of value, particularly those working at the interfaces between analytical/biochemical and molecular biology, as well as the immunological sciences While new developments constantly xiv PREFACE occur, we believe these two volumes provide a solid foundation on which researchers can assess the most recent advances We feel that biologists are working during a truly revolutionary period in which information available for the analysis of biomacromolecular structure and quantitation will provide new insights into fundamental processes We hope these volumes aid readers in advancing significantly their research capabilities WILLIAM S HANCOCK BARRY g KARGER C o n t r i b u t o r s to V o l u m e Article numbers arc in parentheses following the names of contributors Affiliations listed are current MARIE-ISABEL AGUILAR (]), Department of Biochemistry and Centre for Bioprocess Technology, Monash University, Clayton, Victoria 3168, Australia J UP,El) BANKS, JR (21), Analytica of" Branjbrd, Inc., Branford, Connecticut 06405 RONALD C BEAVIS (22), Department (~f Chemistry and Pharmacology, Skirball Institute, New York University, New York, New York 10016 BRUCE W BIRREN (11), Division of Biology, California Institute of Technology, Pasadena, California 91125 PE'I'I~ BO~'EK (17), Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, CZ-611 42 Brno, Czech Republic RICHARD M CAPmOH (20), Analytical Chemistry Center and Department of Biochemistry and Molecular Bioh)gy, University of Texas Medical School, Houston, Texas 77030 BmA~ T CHAIT (22), Laboratory for Mass Spectrometry and Gaseous Ion Chemistry', The Rockefeller University, New York, New York 10021 MARCELLA CHIARI (10), Institute of Hormone Chemistry, National Research Council, Mihrn 20133, ltaly GARGI CHOUDIIARY (3), Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520 BRUCE JON COMPTON (15), Autolmnnrne Inc., Lexington, Massachusetts 02173 MER('EDES DE FRUTOS (4, 6), lnstituto de Quirnica Organica, General y Ferrnentaciones lndustriales (C.S.LC.), 28006 MadrM, Spain Gt/v DROUIN (12), Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5 PE~I~ GE~AUER (17), Institute of' Analytical Chemistry, Academy of' Sciences of the Czech Relmblic, CZ-611 42 Brno, Czech Republic CECmIA G R n (10), Institute of Advanced Biomedical Technologies, National Research Council, Milan, Italy ME'I'TE GRONVALD (15), Department o( Chemistry and Chemical Engineering, The Engineering Academy of Denmark, TIC, 7058, A 892036 Copenhagen, Denmark MIL'LON T W HEARN (1), Department of Biochemistry and Centre for Bioprocess Technology, Monash Univet~'ity, Clayton, Vittoria 3168, Australia STKLLAN HJERTI~N (13), Department of Biochemistry, Uppsala University, Uppsala, Sweden CSABA HORV~,TH (3), Department (~f Chemical Engineering, Yale University, New Haven, Connecticut 06520 IAN JARDINE (23), Finnigan MAT, San Jose, California 95134 JAMES W JORGENSON (18), Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599 LUDMILA KRIVANKOVA(17), Institute of Analytical Chernistrv, Academy (2.[Sciences of the Czech Republic, CZ-611 42 Brno, Czech Rel?ublic BARRY L KARGER (2), Department of Chemistry, Barnett lnstitltte, Northeastern University, Boston, Massachusetts" 0211.5 IRA S KRtILL (8), Department (?r Chemistry, Northeastern University, Boston, Massachusetts 02115 ERIC' LAI (11), Department of Pharrnacology, University of North Cklrolina, Chapel Hill, North Carolina 27599 JOHN P LARMANN, JR (18), Department of Chemistry, University (~t' North Carolina, Chapel Hill, North Carolina 27599 THOMAS T LEE (19), Department of Chernistry, Stanfbrd Universitv, Stanfbrd, Cal([ornia 95305 x CONTRIBUTORS TO VOLUME 270 ANTHONY V LEMMO (18), Department qf' GIRARD P ROZIN(I (9) Waldbronn Ana(vti- Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599 BARBARA D LIPES (l 1), Department of Pharmacology, University qf North Carolina, Chapel Hill, North Carolina 27599 NORlO MAISUBARA (14), Faculty ().['Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, lapan PASCAL MAYER (12), Department (~[Biology, UniversiO; of Ottawa, Ottawa, Ontario, Canada KIN 6N5 JEFF MAZZEO (8), Waters Chromatography Division, Millipore Corporation, Milfi)rd, Massachusetts 01757 ROHIN MHATRE (8), PerSeptive Biosystenzs, Inc., Framingham, Massachusetts 01701 SlAN MI('INSKI (15), Washington State University, Pullman, Washington 99164 AI.vIN W MOORE JR (18), Department q[ Chemistry, University o[" North Carolina, Chapel Hill, North Carolina 27599 MILoS V NOVOTNY(5), Department qfChemistry, bldiana UniversiO', Bloomington, lndiana 47405 SANDEEP K PAl.IWAL (4, 6), SyStemix Inc., Palo Alto, Califi)rnia 94304 FRED E RF(INIEP, (4, 6), Departnlent of Chemistry, Purdue Universitv, Lafilyette, hldiana 47906 PIER GIORGIO RIGIIFTH (i0), Faculty of Pharmacy and Del)artment of Biomedical Sciences and Technologies, Univers,ity qfl Mihm, Milan 20133, lmly ROBERTO RODRI(}t;Ez-DIAz (16), Bio-Rad Laboratories, Hercules, Cal([brnia 94547 cal Division, ttewlett Packard GmbH, D76337 Waldbronn, Germany JAE (7 SCIIWARTZ (23) Finnigan MAT, San Jose, Cal([~)rnia 95134 WII.IIAM E SEIFER'I, Iv, (20), Ana(y, tical Chemist O' Cenwr, University of Texas Medical School, ilouston, Texas 770.t0 GARY W SKATER (12), Department of Physics, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5 LLOYD R SNYI)I'.P, (7), LC Resources, hie., Orinda, Cal(fbrnia 94563 MI('H,'XEt_ SzuI.C" (8), Quality Control R&D Laboratory, Biogen Corporation, Cambridge, Massachusetts 02142 Snn(;ERU TERABE (14), Faculty of Science, Hinteji Institute ()f Technology, Kantigori, Hyogo 678-12, lapin TIM WEaR (16), Bio-Rad Laboratories, ltercules, California 94547 CRAn(; M WHH'EHOt:Sl (21), Ana@tica o[ Branford, Inc., Bran ford, Connecticut 06405 JANET C WRESTLER (11), Department oj' Pharmacology, Universi O, of North Carolina, Chapel Hill, North Carolina 27599 SInAw-LIN WtI (2), Deparmlent of Ana(y'tical Chenlistry, Genenteeh, Inc., South San Francisco, Califi)rnia 94080 EI)WAI',D S YEUNG (19), Department of Chenlistrv and Ames LaboratoiT, Iowa State University, Ames, lowa 50011 MIN(;DE ZUu (16), Bio-Rad Laboratories, Hercules, CaliJbrnia 94.547 [11 RP-HPLC OF PEPTIDES AND PROTEINS [1] H i g h - R e s o l u t i o n R e v e r s e d - P h a s e H i g h - P e r f o r m a n c e Liquid Chromatography of Peptides and Proteins By MARIE-ISABEL AGUILAR and MILTON T W HEARN Introduction Reversed-phase high-performance liquid chromatography (RP-HPLC) has become a commonly used method for the analysis and purification of peptides and proteins ~-3 The extraordinary popularity of RP-HPLC can be attributed to a number of factors, including the excellent resolution that can be achieved for closely related as well as structurally disparate substances under a large variety of chromatographic conditions; the experimental ease with which chromatographic selectivity can be manipulated through changes in mobile phase composition; the generally high recoveries, even at ultramicroanalytical levels; the excellent reproducibility of repetitive separations carried out over long periods of time, due in part to the stability of the various sorbents under many mobile phase conditions; the high productivity in terms of cost parameters; and the potential., which is only now being addressed, for the evaluation of different physicochemical aspects of solute-eluent or solute-hydrophobic sorbent interactions and assessment of their structural consequences from chromatographic data The RP-HPLC experimental system usually comprises an n-alkylsilicabased sorbent from which peptides or proteins are eluted with gradients of increasing concentration of an organic solvent such as acetonitrile containing an ionic modifier, e.g., trifluoroacetic acid (TFA) With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated and low picomolar amounts of resolved components can be collected Separations can be easily manipulated by changing the gradient slope, temperature, ionic modifier, or the organic solvent composition The technique is equally applicable to the analysis of enzymatically derived mixtures of peptides and also for the analysis of synthetically derived peptides An example of the high-resolution analysis of a tryptic digest of bovine growth hormone is shown in Fig Figure demonstrates the rapid M T W H e a r n (ed.), " H P L C of Proteins, Peptides and P o l y n u c l e o t i d e s - - C o n t e m p o r a r y Topics and Applications." VCH, Deerfield, FL, 1991 K M Gooding and F E Regnier (eds.), " H P L C of Biological Macromotecules: Methods and Applications." Marcel Dekker, New York, 1990 C T Mant and R S H e d g e s (eds.), " H P L C of Peptides and Proteins: Separation, Analysis and Conformation." C R C Press, Boca Raton, FL, 1991 METHODS IN ENZYMOLOGY.VOL 270 Copyright ,~t~1996by AcademicPress, Inc All rights of reproduction in any form reserved LIQUID CHROMATOGRAPHY [1] E e6 U e- e~ < t C I I I 15 30 45 Time (min) FIG Reversed-phase chromatographic prolile of a tryptic digest of bovine growth hormone on an n-octadecylsilica sorbent, particle diameter 5/xm, average pore size 30 nm, packed into a 25 cm × 4.6 mm i.d column Gradient elution was carried out from to 50% acetonitrile in 0.1% TFA over 60 at a flow rate of ml/min Detection was at 215 nm (From A J Round, M I Aguilar, and M T W Hearn, unpublished results, 1995.) and highly selective separation that can be achieved with tryptic digests of proteins, using RP-HPLC as part of the quality control or structure determination of a recombinant or natural protein The chromatographic separation shown in Fig was obtained with an octadecylsilica (C~s) stationary phase packed in a column of dimensions 25 cm (length) × 0.46 cm (i.d.) Separated components can be directly subjected to further analysis such as automated Edman sequencing or electrospray mass spectroscopy For the purification of synthetically derived peptides, the crude synthetic product is typically separated on an analytical scale to assess the complexity of the mixture This step is usually followed by large-scale purification and collection of the product, with an aliquot of the purified sample then subjected to further chromatography under different RP-HPLC conditions or another HPLC mode to check for homogeneity Finally, the isolation [11 RP-HPLC OF PEPTIDES AND PROTEINS and analysis of many proteins can also be achieved using high-resolution RP-HPLC techniques In these cases, the influence of protein conformation, subunit assembly, and extent of microheterogeneity becomes an important consideration in the achievement of a high resolution separation and recovery of the active substance by RP-HPLC techniques Nevertheless, RPHPLC methods can form an integral part of the successful isolation of proteins in their native structure, as has been shown, for example, in the purification of transforming growth factor c