high resolution separation and analysis of biological macromolecules, part b

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 vanishingly 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 xiii xiv 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 HC isotopes and often equals that achieved with the use of 32p or 12~I 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 XV 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 xvi 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 L KARGER C o n t r i b u t o r s to V o l u m e Arliclc n u m b e r s art: ill parcnlficscs Iollowing tile haines o l contributols Affiliations listed arc current SAMY ABDEI.-BAKY (21), BASF Corporation, Agricuhural Prodzwts" Center, Research Triangle Park, North Carolina 27709 J()l IN FRENZ (20), Department of Manufacturing Sciences, Genentech, htc., South San Francisco, Ualifornia 94080 KARIMA',2 At.LAM (21), Webb Technical Group, Raleigh, North (klrolina 27612 MICHAEl GIDDINGS (10), Del?artment of Chemisto,, UniversiO' q/' Wisconsin, Madison, Wisconsin 53706 A APH:EL (17) Hewlett Packard Laboratories, Palo Aho, CaliJbrnia 94304 NEBOJSA AVDAI.OVIC (7), Dionex Corporalion, Sunnyvale, California 94088 Lot !ISE3~rEJ BASA (6), Genentech, hw., South San Francisco, Cal!fi?rnia 94080 JAN BERKA (13), Barnett hrstitute, Northeastern University, Boston, Massachusetts 02115 ROBERT L BP.UMLEY,JR (10), GeneSys, lnc., Mazomanie, Wisconsin 53560 EI~.I(7C BI JXTON (10), Department o f Chemisfly, University o f Wisconsin, Madison, Wisconsin 53706 J CHAKH (17), Hewlett Packard Laboratories, Palo Alto, California 94304 STEPIII N CHAN (l 6), Mass Spectrome.y Resource, Boston University Medical Center, Boston, Massachusetts 02118 ROSANNr: C CHLOUPEK (2) Genentech, Inc South San Francisco, Cal(fi)rnia 94080 JOSLPH M COP.BHW (8), Department of Cardiothoracic Sttrgery, National Heart and Lung Institute, Imperial College, Heart Science Centre, ttarefiehl Hospital, Harefield, Middlesex UB9 6JH, United Kingdom MICHAEl J DUNN (8), Department o f 6klrdiothoracic Surgery, Natiomd tteart and Lung Instit.te, hnperial College, Heart Science Centre, ttarefiehl Hospital, tlarefield, Middie.sex UB9 6JH, United Kingdom FRANllSEK FORE1 (13) Barnett hJstitute, Northeastern Universio, Boston, Massaclmsetts 02115 ROC;ER W GIESE (21), Barnett Institute and Bouve College, Northeastern University, Boston, Massachusetts 02115 BErH L GIIJEcE-CASTRO (18) Protein ChemistlT Deparmwnt, Genentech, htc., South San Francisco, California 94080 DAVID R GOODLS3q (19) Chemical Methods and Separations Group, Chemical Sciences Department, Pacific Northwest Laboratory, Richhmd, Washington 99352 A W GUZZETTA (17) Scios Nova, htc., Mountain View, Cal(fornia 94043 Wn.I.IAM S HANCOCK (17), ttewlett Packard Laboratories, Palo Aho, Cal([-brnia 94304 ROBERT S HOD~3ES (1), Deparmzent of Biochemist O' and the Medical Research CozmciL Group in Protein Structure and Fnnction, University of Alberto, Edmonton, Alberto T6G 2H7, Canada EDWARD R HOFF (2), Genentech, Inc., South San Francisco, Cal(lornia 94080 SIEVEN A HOVSTADLER (19) Chemical Methods attd Separations Group, Chemical Sciences Department, Pacific Northwest Laborat(n T, Richland, Washington 99352 L J JANIS (4) Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 D.u;Ro Joslc (5), Octapharma Pharmazeutika Produktionsges.m.b.tt, Research attd Development Department, A-1100 Wien, A stria X CONTRIBUTORS TO VOLUME 271 BARRY L KARGER (13), Department of Chemistry, Barnett Institute, Northeastern University, Boston, Massachusetts 02115 KEN-ICHI KASAI (9), Department of Biological Chemistry, Faculty of Pharmaceutical &iences, Teikyo University, Sagamiko, Kanagawa 199-01 Japan P M KOVACH (4), Lilly Research Labora- tories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 TERRY D LEE (3), City qf Hope, Division o[" InTmunology, Beckman Research Institute, Duarte, Cal(fornia 91010 COLIN T MANT ( 1), Department of Biochem- istt:V and Group in University T6G 2H7 the Medical Research Council, Protein Structure and Fanction, of Alberta, Edmonton, Alberta Canada MICHAEL MAR('HBANKS (10), Hazleton Wis- consin, Mc., Madison, Wisconsin 53704 RANDY M McCORMICK (7), Seurat Analytical Systems, Sunnyvale, Calijbrnia 94089 T M'TIMKt:I.U (17), Berlex Biosciences, Bris- bane, Cal({brnia 94005 MILOS V NOVOINY (14), Department of Chemistry, Indiana University, Bloomington, Indiana 47405 E PUNGOR, JR (17), Berlex Biosciences, Bris'- bane, Cal(&rnia 94005 BRU(E B REINHOLD (16), Mass Spectrometry Resource, Boston University Medical Center, Boston, Massachusetts 02118 VERNON N REINHOLD (~6), Mass Spectrome- try Resource, Boston University Medical Center, Boston, Massachusetts 02118 EUGENE C RI(KARD (11), Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285 R M RIOC~IN (4), Lilly Research Labora- tories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, lndiana 46285 MANASl SAHA (21), BASF Corporation, Ag- ricultural Products Center, Research Triangle Park, North Carolina 27709 KIYOIIIF() SI]IMURA (9), Department of Bio- logical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo Universi(v, Sagamiko, Kanagawa 199-01 Japan City of Hope, Division qf Immunology, Beckman Research Institute, Duarte, Cal(fornia 91010 JOHN E SHIVELY (3), LLOYD M SMrrH (10), Department of Chemis- try, University of Wisconsin, Madison, Wisconsin 53706 RICHARD D SMITH (19), Environmental Mo- lecular Sciences Laboratory, Pacific Northwest National Laboratory, Richhmd, Washington 99352 C SOt!DERS (17), Berh'x Biosciences, Bris- bane, California 94005 MICHAEL W SPELLMAN (6), Genentech, Inc., South San Francisco, Cal(fornia 94080 JolIg T S1UI.TS (18) Protein Chemistry De- partment, Genentech, Inc., South San Francisco, Cal(fbrnia 94080 KRISTINE M SWIDEREK (3), City qfHope, Di- vision of Immunology, Beckman Research Institute, Duarte, California 91010 GI,EN TESIIIMA (12), Department of Analyti- cal Chemistry, Genentech, Inc., South San I@ancisco, California 94080 JAMES R THAYER (7), Dionex Corporation, Sunnyvale, Cal(f'ornia 94088 XIN('HUN TONG (10), Department of Chemis- try, University of Wisconsin, Madison, Wisconsin 53706 JOIIN K TOWNS (4, 11 ), Lilly Research Labo- ratories, Eli Lilly and Company, Lil(v Corporate Center, Indianapolis, Indiana 46285 R REID TOWNSEND (6), Department of Phar- maceutical Chemistrv, University of CaliJornia at San Francisco, San Francisco, California 94143 HAROI_D R UDSE IH (19), Chemical Methods and Separations Group, Chemical Sciences Department, Pactfic Northwest Laboratocv, Richland, Washington 99352 CONTRIBUTORS TO VOLUME 271 xi J():-, H WAHL (19), Chemical Methods and JoHr,, R YA-~ES (15), Department 0[ Molectt- Separations Group, Chemical Sciences Department, Pacific Northwest Laboratot3,, Richland, Washington 99352 SHIAw-LIN W u (12), Department of Analytical Chemistry, Genentech Inc., South Sail Francisco, Cal(fornia 94080 lar Biotechnology, School of Medicine, University of Washington, Seattle, Washington 98195 KAIRIN ZHI.INr.~I~J', (5), Virchow-Klinikum der ttumbold Universit6t, k2~perimentelle CJtirurgie, 13353 Berlin, Germany [1] ANALYSIS OF PEPTIDES BY HPLC [1] A n a l y s i s o f P e p t i d e s b y H i g h - P e r f o r m a n c e Liquid Chromatography By COLIN T MANT and ROBERT S HODGES I Introduction A Focus Even the most superficial perusal of the literature for the purpose of reviewing high-performance liquid chromatography (HPLC) separations of peptides quickly reveals that shortage of relevant material is certainly not a problem This is due primarily to the tremendous development of high-performance chromatographic techniques in the past few years, in terms of scale, instrumentation, and column packings In addition, there is an almost bewildering variety of mobile phases employed by various researchers for specific applications in all major modes of HPLC employed for peptide separations This chapter is aimed at laboratory-based researchers, both beginners and more experienced chromatographers, who wish to learn about peptide applications in HPLC Thus, standard analytical applications in HPLC of peptides will be stressed, as opposed to micro- or preparative-scale chromatography Only nonspecialized columns, mobile phases, and instrumentation readily available and easily employed by the researcher are described in detail In addition, through the use of peptide standards specifically designed for HPLC, the researcher is introduced to standard operating conditions that should first be attempted in the separation of a peptide mixture B Char_acterization of Peptides The distinction between a peptide, polypeptide, or protein, in terms of the number of peptide residues they contain, is somewhat arbitrary However, peptides are usually defined as containing 50 amino acid residues or less Although molecules containing more than 50 residues usually have a stable 3-dimensional structure in solution, and are referred to as proteins, conformation can be an important factor in peptides as well as proteins Secondary structure, e.g., a helix, is generally absent even under benign conditions for small peptides (up to - residues); however, the potential for a defined secondary or tertiary structure increases with increasing peptide length and, for peptides containing more than 20-35 residues, folding ]MKTltOI)S IN KNZYMOLO(JY, VOL 271 Copyright ¢ > Ig96 by Academic Picss Inc ~ All rigllts ot reproduction in any lore1 reserved L~OUI D CHROMATOGRAPHY [ 1] of the peptide chain to internalize nonpolar residues is likely to become an increasingly important conformational feature In addition, the presence of disulfide bridge(s) would be expected to affect peptide conformation and, thus, the retention behavior of a peptide in H P L C may differ from that in the fully reduced state L Thus, conformation should always be a consideration when choosing the conditions for chromatography C Peptide Detection Peptide bonds absorb light strongly in the far ultraviolet (