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

635 Pages·1996·10.913 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 wmishingly
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
xii ECAFERP
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 C41 isotopes and often equals
that achieved with the use of p23 or I521 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 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
xiv 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. HANCOCK
BARRY g. KARGER
Contributors to Volume 270
Article numbers arc in parentheses following the names of contributors.
Affiliations listed are current.
MARIE-ISABEL AGUILAR (]), Department of Czech Relmblic, CZ-611 42 Brno, Czech
Biochemistry and Centre for Bioprocess Republic
Technology, Monash University, Clayton, CECmIA GRn (10), Institute of Advanced
Victoria 3168, Australia Biomedical Technologies, National Re-
J. ,PU El) JR. BANKS, (21), Analytica "fo Bran- search Council, Milan, Italy
jbrd, Inc., Branford, Connecticut 06405 ME'I'TE GRONVALD (15), Department o(
DLANOR C. BEAVIS (22), Department f~( Chemistry and Chemical Engineering, The
Chemistry and Pharmacology, Skirball In- Engineering Academy of Denmark, ,CIT
stitute, New York University, New York, ,8507 A 892036 Copenhagen, Denmark
New York 10016 L'LIM NO T. W. HEARN (1), Department of Bio-
BRUCE W. BIRREN (11), Division of Biology, chemistry and Centre for Bioprocess Tech-
California Institute of Technology, Pasa- nology, Monash ,yti'~tevinU Clayton, Vitto-
dena, California 91125 air 3168, Australia
PE'I'I~ BO~'EK (17), Institute of Analytical NALLKTS N~ITREJH (13), Department of Bio-
Chemistry, Academy of Sciences of the chemistry, Uppsala University, Uppsala,
Czech Republic, CZ-611 42 Brno, Czech Sweden
Republic CSABA HT,~VROH (3), Department f~( Chemi-
DRAHCIR M. CAPmOH (20), Analytical Chem- cal Engineering, Yale University, New Ha-
istry Center and Department of Biochemis- ven, Connecticut 06520
try and Molecular Bioh)gy, University of NAI JARDINE (23), Finnigan MAT, San Jose,
Texas Medical School, Houston, Texas California 95134
03077 JAMES W. NOSNEGROJ (18), Department of
BmA~ T. CHAIT (22), Laboratory for Mass Chemistry, University of North Carolina,
Spectrometry and Gaseous Ion Chemistry', Chapel Hill, North Carolina 27599
The Rockefeller University, New York, New AVOKNAVIRK ALIMDUL (17), Institute of Ana-
York 12001 lytical Chernistrv, Academy [.2( Sciences of
ALLECRAM CHIARI (10), Institute of Hormone the Czech Republic, CZ-611 24 Brno,
Chemistry, National Research Council, Mi- Czech Rel?ublic
nrh 20133, ltaly BARRY L. KARGER (2), Department of Chem-
GARGI CHOUDIIARY (3), Department of istry, Barnett lnstitltte, Northeastern Univer-
Chemical Engineering, Yale University, sity, Boston, "sttesuhcassaM 5.1120
New Haven, Connecticut 06520 ARI S. KRtILL (8), Department r?( Chemistry,
BRUCE NOTPM ONCOJ (15), Autolmnnrne Inc., Northeastern University, Boston, Massa-
Lexington, Massachusetts 02173 chusetts 02115
MER('EDES ED FRUTOS (4, 6), lnstituto de 'CIRE LAI (11), Department of Pharrnacology,
Quirnica Organica, General y Ferrnentaci- University of North Cklrolina, Chapel Hill,
ones lndustriales (C.S.LC.), 28006 Ma- North Carolina 27599
drM, Spain JOHN P. ,NNAMRAL JR. (18), Department of
Gt/v DROUIN (12), Department of Biology, Chemistry, University 't~( North Carolina,
University of Ottawa, Ottawa, Ontario, Chapel Hill, North Carolina 27599
Canada K1N 6N5 SAMOHT T. LEE (19), Department of Chernis-
~I~EP GE~AUER (17), Institute of' Analytical try, Stanfbrd Universitv, Stanfbrd, Cal([or-
Chemistry, Academy of' Sciences of the nia 95305
x CONTRIBUTORS TO VOLUME 270
YNOHTNA V. LEMMO (18), Department qf' GIRARD P. ROZIN(I (9). Waldbronn Ana(vti-
Chemistry, University of North Carolina, cal Division, ttewlett Packard GmbH,
Chapel Hill, North Carolina 27599 D76337 Waldbronn, Germany
ARABRAB D. LIPES (l 1), Department of Phar- JAE .7( ZTRAWIICS (23). Finnigan MAT, San
macology, University qf North Carolina, Jose, ainr)~(laC 95134
Chapel Hill, North Carolina 27599 WII.IIAM E. SEIFER'I, Iv,. (20), Ana(y, tical
NORlO MAISUBARA (14), Faculty ,ecneicS'.)( Chemist 'O Cenwr, University of Texas Med-
Himeji Institute of Technology, Kamigori, ical School, ilouston, Texas 770.t0
Hyogo 678-12, .lapan GARY W. RETAKS (12), Department of Phys-
LACSAP MAYER (12), Department (~Biology, ics, University of Ottawa, Ottawa, Ontario,
;OisrevinU of Ottawa, Ottawa, Ontario, Canada K1N 6N5
Canada KIN 6N5 DYOLL R. ,P.'I)IYNS (7), LC Resources, hie.,
JEFF MAZZEO (8), Waters Chromatography Orinda, Cal(fbrnia 94563
Division, Millipore Corporation, Milfi)rd, _tEX',H'(IM SzuI.C" (8), Quality Control R&D
Massachusetts 01757 Laboratory, Biogen Corporation, Cam-
ROHIN MHATRE (8), PerSeptive Biosystenzs, bridge, Massachusetts 02142
Inc., Framingham, Massachusetts 01701 nS n(;ERU TERABE (14), Faculty of Science, Hi-
SlAN MI('INSKI (15), Washington State Univer- nteji Institute ()f Technology, Kantigori, Hy-
sity, Pullman, Washington 99164 ogo 678-12, .lapin
AI.vIN W. MOORE. JR. (18), Department q MIT WEaR (16), Bio-Rad Laboratories, lter-
Chemistry, University "o North Carolina, cules, California 94547
Chapel Hill, North Carolina 27599 CRAn(; M. WHH'EHOt:Sl (21), Ana@tica o
MILoS V. YNTOVON (5), Department qfChem- Bran ford, Inc., Bran ford, Connecticut
istry, bldiana UniversiO', Bloomington, ln- 50460
diana 47405 JANET C. WRESTLER (11), Department oj'
PEEDNAS K. PAl.IWAL (4, 6), SyStemix Inc., Pharmacology, Universi ,O of North Caro-
Palo Alto, Califi)rnia 94304 lina, Chapel Hill, North Carolina 27599
FRED E. RF(INIEP, (4, 6), Departnlent of SInAw-LIN WtI (2), Deparmlent of Ana(y'tical
Chemistry, Purdue Universitv, Lafilyette, Chenlistry, Genenteeh, Inc., South San
hldiana 47906 Francisco, Califi)rnia 94080
PIER GIORGIO RIGIIFTH (i0), Faculty of D,'IAW)IE S. YEUNG (19), Department of
Pharmacy and Del)artment of Biomedical Chenlistrv and Ames LaboratoiT, Iowa
Sciences and Technologies, ,srevinU ity qfl State University, Ames, lowa 50011
Mihm, Milan 20133, lmly MIN(;DE ZUu (16), Bio-Rad Laboratories,
ROBERTO RODRI(}t;Ez-DIAz (16), Bio-Rad Hercules, CaliJbrnia 745.49
Laboratories, Hercules, Cal(brnia 94547
11[ RP-HPLC OF PEPTIDES AND PROTEINS 3
[1] High-Resolution Reversed-Phase High-Performance
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 ofc hromatographic conditions; the experi-
mental 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 repeti-
tive 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-alkylsilica-
based sorbent from which peptides or proteins are eluted with gradients
of increasing concentration of an organic solvent such as acetonitrile con-
taining 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 gradi-
ent 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 pep-
tides. An example of the high-resolution analysis of a tryptic digest of
bovine growth hormone is shown in Fig. .1 Figure 1 demonstrates the rapid
1 M. T. W. Hearn (ed.), "HPLC of Proteins, Peptides and Polynucleotides--Contemporary
Topics and Applications." VCH, Deerfield, FL, 1991.
2 K. M. Gooding and F. E. Regnier (eds.), "HPLC of Biological Macromotecules: Methods
and Applications." Marcel Dekker, New York, 1990.
3 C. T. Mant and R. S. Hedges (eds.), "HPLC of Peptides and Proteins: Separation, Analysis
and Conformation." CRC Press, Boca Raton, FL, 1991.
thgirypoC ~t~, 6991 yb cimedacA Press, .cnI
SDOHTEM NI .YGOLOMYZNE .LOV 072 llA of rights noitcudorper ni yna form .devreser
4 LIQUID CHROMATOGRAPHY [1]
7
E
e-
6 8
5
U
e-
C
e~
<
t I I I
0 51 30 45
Time (min)
FIG. .1 Reversed-phase chromatographic prolile of a tryptic digest of bovine growth hor-
mone 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 0 to 50% acetonitrile
in 0.1% TFA over 60 min at a flow rate of 1 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. 1 was obtained with an octadecylsilica (C~s) station-
ary 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 5
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 recov-
ery of the active substance by RP-HPLC techniques. Nevertheless, RP-
HPLC 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 4 inhibin, 5 thyroid-stimulating
hormone) growth hormone] and insulin. 8 However, it should be noted
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in RP-HPLC either in terms of recovered mass or the loss of activity.
The success of RP-HPLC, which is illustrated by the selected examples
in Table 9 I, o~ is also due to the ability of this technique to probe the
hydrophobic surface topography of a biopolymer. This specificity arises
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6 LIQUID CHROMATOGRAPHY ] 1 [
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