High Resolution Separation and Analysis of Biological Macromolecules: Fundamentals Part B

571 Pages·1996·9.997 MB·English

by  Abelson J.N., Hancock W.S., Simon M.I. (eds.)

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 mac-
romolecules 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 be-
ing 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 disadvan-
tages 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 re-
actions.
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 DNA 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
iiix
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 separa-
tion-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 frag-
ments 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 de-
termining 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 mea-
surements are frequently complemented by more traditional approaches,
e.g., N-terminal sequencing of proteins or the Sanger method for the se-
quencing of oligonucleotides. Furthermore, a recent trend is to follow
kinetically the enzymatic degradation of a macromolecule (e.g., carboxy-
peptidase). 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 (MALDI-TOF), individual resi-
dues that have been cleaved (e.g., amino acids) can be determined.
As well as producing detailed chemical information on the macromole-
cule, many of these methods also have the advantage of a high degree of
mass sensitivity since new instrumentation, such as MALDI-TOF or capil-
lary 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 p23 or I~21 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 pro-
vide 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 meth-
odological 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 Vol-
ume 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 spectrom-
etry. 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 infor-
mation.
The focus of the LC section is on updating the most significant new
approaches to biomolecular analysis. LC has been covered in recent vol-
umes of this series, therefore these volumes concentrate on relevant applica-
tions that allow for automation, greater speed of analysis, or higher separa-
tion 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 mea-
surements 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 ECAFERP
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 avail-
able 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 KCOCNAH
BARRY L. KARGER
Contributors to Volume 271
Arliclc numbers art: ill parcnlficscs Iollowing tile haines ol contributols.
Affiliations listed arc current.
YMAS ABDEI.-BAKY (21), BASF Corporation, J()l NI FRENZ (20), Department of Manufactur-
Agricuhural Prodzwts" Center, Research Tri- ing Sciences, Genentech, htc., South San
angle Park, North Carolina 27709 Francisco, Ualifornia 94080
2,'AMIRAK At.LAM (21), Webb Technical MICHAEl GIDDINGS (10), Del?artment of
Group, Raleigh, North (klrolina 27612 Chemisto,, Universi 'O q/' Wisconsin, Madi-
A. APH:EL (17). Hewlett Packard Labora- son, Wisconsin 53706
tories, Palo Aho, CaliJbrnia 94304 RE;COR W. GIESE (21), Barnett Institute and
ASJOBEN AVDAI.OVIC (7), Dionex Corpora- Bouve College, Northeastern University,
lion, Sunnyvale, California 94088 Boston, Massachusetts 02115
Lot Er~3ESI! J. BASA (6), Genentech, hw., South BErH L. GIIJEcE-CASTRO (18). Protein
San Francisco, Cal!fi?rnia 94080 Chemistl T Deparmwnt, Genentech, htc.,
JAN BERKA (13), Barnett hrstitute, Northeast- South San Francisco, California 94080
ern University, Boston, Massachusetts
DAVID R. GOODLS3q (19). Chemical Methods
02115
and Separations Group, Chemical Sciences
TREBOR L. ,YELMU.PB JR. (10), GeneSys, lnc., Department, Pacific Northwest Laboratory,
Mazomanie, Wisconsin 53560 Richhmd, Washington 99352
7(I.~IE C. BI NOTXJ (10), Department of Chemis- A. W. GUZZETTA (17). Scios Nova, htc.,
fly, University of Wisconsin, Madison, Wis- Mountain View, Cal(fornia 94043
consin 53706
Wn.I.IAM S. KCOCNAH (17), ttewlett Packard
J. CHAKH (17), Hewlett Packard Labora-
Laboratories, Palo Aho, Cal(-brnia 94304
tories, Palo Alto, California 94304
ROBERT S. SE3~DOH (1), Deparmzent of Bio-
IIIPETS N CHAN (l 6), Mass Spectrome.y Re-
chemist O' and the Medical Research Cozm-
source, Boston University Medical Center,
Lic Group in Protein Structure and Fnnc-
Boston, Massachusetts 02118
tion, University of Alberto, Edmonton,
:rNNASOR C. CHLOUPEK (2). Genentech, Inc. Alberto T6G 2H7, Canada
South San Francisco, Cal(fi)rnia 94080
EDWARD R. HOFF (2), Genentech, Inc., South
JOSLPH M. COP.BHW (8), Department of
San Francisco, Cal(lornia 94080
Cardiothoracic Sttrgery, National Heart and
Lung Institute, Imperial College, Heart Sci- SIEVEN A. HOVSTADLER (19). Chemical
ence Centre, ttarefiehl Hospital, Harefield, Methods attd Separations Group, Chemical
Middlesex UB9 6JH, United Kingdom Sciences Department, Pacific Northwest
Laborat(n T, Richland, Washington 99352
MICHAEl. J. DUNN (8), Department of 6klrdio-
thoracic Surgery, Natiomd tteart and Lung L. J. JANIS (4). Lilly Research Laboratories,
Instit.te, hnperial College, Heart Science Eli Lilly and Company, Lilly Corporate
Centre, ttarefiehl Hospital, tlarefield, Mid- Center, Indianapolis, Indiana 46285
die.sex UB9 6JH, United Kingdom D.u;Ro Joslc (5), Octapharma Pharmazeutika
FRANllSEK FORE1 (13). Barnett hJstitute, Produktionsges.m.b.tt, Research attd De-
Northeastern Universio,. Boston, Massa- velopment Department, A-1100 Wien,
clmsetts 02115 A .stria
X CONTRIBUTORS TO VOLUME 271
BARRY L. KARGER (13), Department of KIYOIIIF() SIIMURA (9), Department of Bio-
Chemistry, Barnett Institute, Northeastern logical Chemistry, Faculty of Pharmaceuti-
University, Boston, Massachusetts 02115 cal Sciences, Teikyo Universi(v, Sagamiko,
Kanagawa 199-01 Japan
IHCI-NEK KASAI (9), Department of Biological
Chemistry, Faculty of Pharmaceutical &i- NHOJ E. YLEVIHS (3), City of Hope, Division
ences, Teikyo University, Sagamiko, Kana- qf Immunology, Beckman Research Insti-
gawa 199-01 Japan tute, Duarte, Cal(fornia 91010
P. M. KOVACH (4), Lilly Research Labora- LLOYD M. SMrrH (10), Department of Chemis-
tories, Eli Lilly and Company, Lilly Corpo-
try, University of Wisconsin, Madison, Wis-
rate Center, Indianapolis, Indiana 46285
consin 53706
TERRY D. LEE (3), City qf Hope, Division "o
RICHARD D. SMITH (19), Environmental Mo-
InTmunology, Beckman Research Institute,
lecular Sciences Laboratory, Pacific North-
Duarte, Cal(fornia 91010
west National Laboratory, Richhmd, Wash-
COLIN T. MANT ( 1 ), Department of Biochem- ington 99352
V:ttsi and the Medical Research Council,
Group in Protein Structure and Fanction, C. SOt!DERS (17), Berh'x Biosciences, Bris-
bane, California 94005
University of Alberta, Edmonton, Alberta
T6G 2H7 Canada MICHAEL W. NAMLLEPS (6), Genentech, Inc.,
LEAHCIM MAR('HBANKS (10), Hazleton Wis- South San Francisco, Cal(fornia 94080
consin, Mc., Madison, Wisconsin 53704
JolIg T. ST.IU1S (18). Protein Chemistry De-
YDNAR M. McCORMICK (7), Seurat Analytical partment, Genentech, Inc., South San Fran-
Systems, Sunnyvale, Calijbrnia 94089 cisco, Cal(fbrnia 94080
T. M'TIMKt:I.U (17), Berlex Biosciences, Bris- ENITSIRK M. SWIDEREK (3), City qfHope, Di-
bane, Cal({brnia 94005 vision of Immunology, Beckman Research
MILOS V. NOVOINY (14), Department of Institute, Duarte, California 91010
Chemistry, Indiana University, Blooming-
GI,EN TESIIIMA (12), Department of Analyti-
ton, Indiana 47405
cal Chemistry, Genentech, Inc., South San
E. PUNGOR, JR. (17), Berlex Biosciences, -'sirB ,ocsicna@I California 94080
bane, Cal(&rnia 94005
JAMES R. THAYER (7), Dionex Corporation,
BRU(E B. DLOHNIER (16), Mass Spectrometry Sunnyvale, Cal(f'ornia 94088
Resource, Boston University Medical Cen-
ter, Boston, Massachusetts 02118 XIN('HUN TONG (10), Department of Chemis-
try, University of Wisconsin, Madison, Wis-
NONREV N. DLOHNIER (~6), Mass Spectrome-
consin 53706
try Resource, Boston University Medical
Center, Boston, Massachusetts 02118 JOIIN K. SNWOT (4, 11 ), Lilly Research Labo-
ratories, Eli Lilly and Company, Lil(v Cor-
EUGENE C. RI(KARD (11), Lilly Research
porate Center, Indianapolis, Indiana 46285
Laboratories, Eli Lilly and Company, Lilly
Corporate Center, Indianapolis, Indiana R. REID TOWNSEND (6), Department of Phar-
58264 maceutical Chemistrv, University of Cali-
R. M. NI~COIR (4), Lilly Research Labora- Jornia at San Francisco, San Francisco, Cal-
tories, Eli Lilly and Company, Lilly Corpo- ifornia 94143
rate Center, Indianapolis, lndiana 46285 HAROI_D R. UDSE IH (19), Chemical Methods
MANASl SAHA (21), BASF Corporation, Ag- and Separations Group, Chemical Sciences
ricultural Products Center, Research Trian- Department, Pactfic Northwest Laboratocv,
gle Park, North Carolina 27709 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 De- lar Biotechnology, School of Medicine, Uni-
partment, Pacific Northwest Laboratot3,, versity of Washington, Seattle, Washing-
Richland, Washington 99352 ton 98195
SHIAw-LIN Wu (12), Department of Analyti- KAIRIN ZHI.INr.~I~J', (5), Virchow-Klinikum
cal Chemistry, Genentech. Inc., South Sail der ttumbold Universit6t, k2~perimentelle
Francisco, Cal(fornia 94080 ,eigruritJC 13353 Berlin, Germany
1
ANALYSIS OF PEPTIDES BY HPLC 3
1 Analysis of Peptides by High-Performance
Liquid Chromatography
By COLIN T. MANT and ROBERT .S SEGDOH
.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 chroma-
tography. Only nonspecialized columns, mobile phases, and instrumenta-
tion 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 noitaziretca_rahC of Peptides
The distinction between a peptide, polypeptide, or protein, in terms of
the number of peptide residues they contain, is somewhat arbitrary. How-
ever, 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 -15 residues); however, the potential
for a defined secondary or tertiary structure increases with increasing pep-
tide length and, for peptides containing more than 20-35 residues, folding
Copyright >~¢ Ig96 by Academic Picss. Inc.
MKTltOI)S IN KNZYMOLO(JY, VOL. 172 All rigllts ot reproduction in any lore1 reserved.
4 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 HPLC 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 (<220 nm),
providing a convenient means of detection (usually between 210 and 220
rim). In addition, the aromatic side chains of tyrosine, phenylalanine, and
tryptophan absorb light in the 250 to 290-nm ultraviolet range. The develop-
ment of multiwavelength detectors, enabling the simultaneous detection of
peptide bond and aromatic side-chain absorbance, has proved of immense
value for the rapid separation and identification of peptides and proteins.
.D Major Modes of HPLC Used ni Peptide Separations
Because amino acids are the fundamental units of peptides, the chro-
matographic behavior of a particular peptide will be determined by the
number and properties (polarity, charge potential) of the residue side chains
it contains. Thus, the major modes of HPLC employed in peptide separa-
tions take advantage of differences in peptide size (size-exclusion HPLC,
or SEC), net charge (ion-exchange HPLC, or IEC), or hydrophobicity
(reversed-phase HPLC, or RP-HPLC; and, to a lesser extent, hydrophobic
interaction chromatography, or HIC). Within these modes, mobile-phase
conditions may be manipulated to maximize the separation potential of a
particular HPLC column.
.E Peptide Sources and Separation "slaoG
HPLC has proved versatile in the isolation of peptides from a wide
variety of sources. The complexity of peptide mixtures will vary widely
depending on the source, because peptides derived from various sources
differ widely in size, net charge, and polarity. In addition, the quantity of
peptides to be isolated will depend on their origin, e.g., peptides obtained
from biological tissues are oftep found only in small quantities, whereas
quantities of peptides obtained from protein cleavage or solid phase synthe-
sis may be considerably larger.
1 .K .K Lce, .J .A ,kcalB dna .R .S ,segdoH ni "HPLC of seditpeP dna :snietorP ,noitarapeS
sisylanA dna Conformation" .C( .T Mant dna .R .S Hodges, eds.), .p .983 CRC ,sserP
acoB Raton, ,LF .1991
1 ANALYSIS OF PEPTIDES BY HPLC 5
As a general rule, the approach to separation must be tailored to the
separation goals, i.e., purification of a single peptide from a complex mixture
(e.g., the purification of a synthetic peptide from synthetic impurities follow-
ing solid phase peptide synthesis) will require a different approach to that
necessary for separating all components of a complex mixture (e.g., peptide
fragments resulting from tryptic cleavage of a protein). The former ap-
proach may only require the application of a single HPLC technique, i.e.,
taking advantage of only one property (size, charge, or polarity) of the
peptide of interest. In contrast, the latter approach will generally require
a combination of separation techniques (SEC, IEC, and RP-HPLC) (multi-
dimensional or multistep HPLC) for efficient separation of all desired
peptides. The reader is directed to Refs. 2-8 for selected practical examples
of approaches to multidimensional HPLC of peptides, a brief review of
which can be found in Ref. .9
.F Peptide HPLC Standards
Common to all peptide applications in HPLC is the need to choose the
correct column(s) and the most suitable mobile phase. The logical approach
to this is the employment of standards, specifically peptide standards, to
monitor the suitability of HPLC columns and conditions. Peptide standards
are best suited for monitoring peptide retention behavior in HPLC, because
it is preferable to use standards that are structurally similar to the sample
of interest. Among other things, peptide standards allow the researcher to
monitor column performance (efficiency, selectivity, and resolution), run-
to-run reproducibility, column aging, instrumentation variations, and the
effect of varying run parameters (e.g., the flow rate in SEC, IEC, and RP-
HPLC or the gradient rate in IEC and RP-HPLC) and temperature. In
addition, and importantly, peptide standards allow the researcher to identify
nonideal peptide retention behavior on a particular HPLC column, as well
as to develop approaches for manipulation or suppression of such nonideal
behavior through changes in the mobile phase.
The value of peptide standards (or other types of standards, depending
on the compounds to be separated) in monitoring peptide retention behav-
H. Mabuchi and H. Nakahashi, .J Chromalogr. 213, 275 (1981).
N. Takahashi, Y. Takahashi, and F. W. Putnam. .J Chromatogr. 266, 511 (1983).
4 C T. Mant and R. S. Hodges, .J Chronmtogr. 326, 349 (1985).
J. Eng., C.-G. Huang. Y.-C. Pan, J. D. Hulmes, and R. S. Yalow, Peptides 8, 165 (1987).
'~ P. Young, T. Wheal. J. Grant, and T. Kearncy, LC-GC 9, 726 (1991).
7 K. Matsuoka. M. Taoka. T. lsobe, T. Okuyama, and Y. Kato..l. Chromatogr. 515, 313 (199(/).
s N. Lundell and K. Markidcs, Chromatographia 34, 369 (I992).
J' C. T. Mant and R. S. Hodges, .I, Liq. Chromatogr. 12, 139 (1989).