Methods in Enzymology, Vol. 369: Combinatorial Chemistry, Part B

566 Pages·2003·7.447 MB·English

by  Guillermo A. Morales, Barry A. Bunin (Eds.)

Preface
Combinatorial chemistry has matured from a field where efforts initially
focused on peptide-based research to become an indispensable research tool
formolecularrecognition,chemical-propertyoptimization,anddrugdiscovery.
Originallyusedasamethodtoprimarilygeneratelargenumbersofmolecules,
combinatorial chemistry has been significantly influenced and integrated with
other important fields such as medicinal chemistry, analytical chemistry, syn-
theticchemistry,robotics,andcomputationalchemistry.
Eventhoughtheinitialfocusofattentionwasprovidinglargernumbersof
molecules with a ‘‘diversity’’ goal in mind, other factors came into play
dependingupontheproblemscientistsweretryingtosolve,suchasbioactivity,
solubility, permeability properties, PK, ADME, toxicity, and patentability.
One can think of combinatorial chemistry and compound screening as an
iterative Darwinian process of divergence and selection. Particularly in drug
discovery, where time is a critical factor to success, combinatorial chemistry
offersthemeanstotestmoremoleculehypothesesinparallel.
We will always be limited to a finite number of molecules that we can
economically synthesize and evaluate. Even with all the advances in automa-
tiontechnologies,combinatorialchemistry,andhigher-throughputscreensthat
improve our ability to rapidly confirm or disprove hypotheses, the synthesis
and screening cycle remains the rate-determining process. Fortunately, we
continue to make great strides forward in the quality and refinement of pre-
dictivealgorithmsandinthebreadthofthetrainingsetsamassedtoaidinthe
drugdiscovery/compoundoptimizationiterativeprocess.
Anyonewhohasoptimizedchemicalreactionsforcombinatoriallibraries
orprocesschemistryknowsfirsthandhowmuchexperimentationisrequiredto
identify optimal conditions. Chemical feasibility is at the heart of small mol-
ecule discovery and chemotype prioritization since it essentially defines what
canandcannotbeanaloged(i.e.,analogability).Althoughanalogabilityisnot
the only driving factor, quite often it is overlooked. For example, when com-
mercially-available compounds or complex natural products are screened, the
leads generated are often dropped because of the difficulty to rapidly analog
themintheleadoptimizationstage.
The desirability of a chemotype is a function of drug-likeness, potency,
novelty, and analogability. A particularly attractive feature of combinatorial
chemistry is that when desirable properties are identified, they can often be
xiii
xiv preface
optimized through second-generation libraries following optimized synthetic
protocols. If this process of exploring truly synthetically accessible chemical
spaces could be automated, then it would open up the exciting possibility of
modelingtheiterativesynthesisandscreeningcycle.
Predicting, or even just mapping, synthetic feasibility is a sleeping giant;
fewpeoplearelooking intoit,andtheramificationsofabreakthroughwould
berevolutionaryforbothchemistryanddrugdiscovery.In-roadstopredicting
(orevenjustmapping)chemicalfeasibilityhavethepotentialtohaveaslarge
an impact on drug discovery as computational models of bioavailability and
drugability.Theseareimportantquestionswherescientistsarenowstartingto
generate a large-enough body of information on high-throughput synthetic
chemistry to begin to more globally understand what is cost-effectively pos-
sible. Within the biopharmaceutical industry, significant investments in new
technologieshavebeenmadeinmolecularbiology,genomics,andproteomics.
However, with the exception of combinatorial chemistry, relatively little has
been done to advance the fundamental nature of chemistry in drug discovery
fromaconceptualperspective.
Now, after having gone through the molecule-generating period where
research institutions have a large historical compound collection and the pro-
liferation of combinatorial chemistry services, the trend is now after making
more targeted-oriented molecular entities also known as ‘‘focused libraries.’’
An important emerging question is: How can one most effectively make the
best possible ‘‘focused libraries’’ to answer very specific research questions,
givenallthepossiblemoleculesonecouldtheoreticallysynthesize?
The first installment in this series (Volume 267, 1996) mostly covered
peptide and peptidomimetic based research with just a few examples of small
molecule libraries. In this volume we have compiled cutting-edge research in
combinatorial chemistry, including divergent areas such as novel analytical
techniques, microwave-assisted synthesis, novel linkers, and synthetic ap-
proachesinbothsolid-phaseandpolymer-assistedsynthesisofpeptides,small
molecules, and heterocyclic systems, as well as the application of these tech-
nologiestooptimizemolecularpropertiesofscientificandcommercialinterest.
Guillermo A. Morales
Barry A. Bunin
METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
John N. Abelson Melvin I. Simon
DIVISIONOFBIOLOGY
CALIFORNIAINSTITUTEOFTECHNOLOGY
PASADENA,CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
Contributors to Volume 369
Articlenumbersareinparenthesesandfollowingthenamesofcontributors.
Affiliationslistedarecurrent.
Fernando Albericio (2), University BalanChenera(24),AmgenInc.,Depart-
of Barcelona, Barcelona Biomedical mentofSmallMoleculeDrugDiscovery,
Research Institute, Barcelona Science One Amgen Center Drive, Thousand
Park, Josep Samitier 1, Barcelona, Oaks,California,91320
08028,Spain
James W. Christensen (5), Advanced
Alessandra Bartolozzi (19), Surface ChemTech Inc., 5609 Fern Valley Road,
Logix, Inc., 50 Soldiers Field Place, Louisville,Kentucky,40228
Brighton,Massachusetts,02135
Andrew P. Combs (12), Incyte Corpo-
Hugues Bienayme´ (24), Chrysalon Mo-
ration,Wilmington,Delaware,19880-0500
lecularResearch,IRC,11AlbertEinstein
Avenue,Villeurbannem,69100,France Scott M. Cowell (16), Department of
Chemistry, University of Arizona,
Sylvie E. Blondelle (18), Torrey Pines
Tucson,Arizona,85721
Institute for Molecular Studies, 3550
General Atomics Court, San Diego, Stefan Dahmen (7), Institut fur Orga-
California,92121
nische Chemie, RWTH Aachen, Pirlet-
Ce´sar Boggiano (18), Torrey Pines Str.1,Aachen,52074,Germany
Institute for Molecular Studies, 3550 Ninh Doan (17), Division of Hematology
General Atomics Court, San Diego,
and Oncology, Department of Internal
California,92121
Medicine,UCDavisCancerCenter,Uni-
Stefan Bra¨se (7), Institut fu¨r Organische versity of California Davis, Sacramento,
Chemie, Universita¨t Karlsruhe (TH), California,95817
Fritz-Haber-Weg 6, Karlsruhe, D-76131,
Roland E. Dolle (8), Senior Director of
Germany
Chemistry, Department of Chemistry,
Andrew M. Bray (3), Mimotopes Pty Adolor Corporation, 700 Pennsylvania
Ltd., 11 Duerdin Street, Clayton, Vic- Drive,Exton,Pennsylvania,19345
toria,3168,Australia
Nicholas Drinnan (14), Alchemia Pty
Wolfgang K.-D. Brill (23), Discovery Ltd., Eight Mile Plains, Queensland
Research Oncology, Pharmacia Italy 4113,Australia
S.p.A,VialePasteur10,Nerviano(MI),
I-20014,Italy Amanda M. Enstrom (17), Division of
Hematology and Oncology, Department
MaxBroadhurst(14),AlchemiaPtyLtd., of Internal Medicine, UC Davis Cancer
EightMilePlains,Queensland4113,Aus- Center, University of California Davis,
tralia Sacramento,California,95817
ix
x contributors to volume 369
Liling Fang (1), ChemRx Division, Dis- RichardHoughten(25),TorreyPinesIn-
coveryPartnersInternational,385Oyster stitute for Molecular Studies, 3550 Gen-
Point Boulevard, Suite 1, South San eral Atomics Court, Room 2-136, San
Francisco,California,94080 Diego,California,92121
Eduard R. Felder (23), Discovery Re- Victor J. Hruby (16), Department of
search Oncology, Pharmacia Italy Chemistry, University of Arizona,
S.p.A., Viale Pasteur 10, Nerviano Tucson,Arizona,85721
(MI),I-20014,Italy
ChristopherHulme(24),AmgenInc.,De-
A´rpa´d Furka (5), Eo¨tvo¨s Lora´nd Univer- partmentofSmallMoleculeDrugDiscov-
sity, Department of Organic Chemistry, ery, One Amgen Center Drive, 29-1-B,
P.O. Box 32, Budapest 112, H-1518, ThousandOaks,California,91320
Hungary
SharonA.Jackson(12),AventisPharma-
A.Ganesan(22),UniversityofSouthamp- ceuticals, 202-206, Bridgewater, New
ton,DepartmentofChemistry,Highfield, Jersey,08807-0800
Southampton,SO171BJ,UnitedKingdom
IanW.James(3),MimotopesPtyLtd.,11
J. Gabriel Garcia (20), 4SC AG, Am Duerdin Street, Clayton, Victoria, 3168,
Klopferspitz 19A, 82152, Martinsried, Australia
Germany
Wyeth Jones (24), Amgen Inc., Depart-
Brian Glass (13), Incyte Corporation, mentofSmallMoleculeDrugDiscovery,
Wilmington,Delaware,19880-0500 OneAmgenCenterDrive,29-1-B,Thou-
sandOaks,California,91320
MatthiasGrathwohl(14),AlchemiaPty
Ltd.,EightMilePlains,Queenland4113, Patrick Jouin (10), CNRS UPR 9023,
Australia CCIPE,141,ruedelaCardonille,Mont-
pellierCedex05,34094,France
Michael J. Grogan (19), Surface Logix,
Inc., 50 Soldiers Field Place, Brighton, C.OliverKappe(11),InstituteofChemis-
Massachusetts,02135 try, Karl-Franzens-University Graz,
Heinrichstrasse28,Graz,A-8010,Austria
Xuyuan Gu (16), Department of
Chemistry, University of Arizona, StevenA.Kates(19),SurfaceLogix,Inc.,
Tuscon,Arizona,85721 50SoldiersFieldPlace,Brighton,Massa-
chusetts,02135
EricHealy(5),AdvancedChemTechInc.,
5609 Fern Valley Road, Louisville, Viktor Krchnˇa´k (6), Torviq, 3251 West
Kentucky,40228 LambertLane,Tuscon,Arizona,85742
Timothy F. Herpin (4), Rhoˆne-Poulenec KitS.Lam(15,17), DivisionofHematol-
Rorer, 500 Arcola Road, Collegeville, ogy and Oncology, Department of In-
Pennsylvania,19426 ternal Medicine, UC Davis Cancer
Center, University of California Davis,
CorneliaE.Hoesl(25),TorreyPinesIn- Sacramento,California,95817
stitute,Room2-136,3550GeneralAtom-
icsCourt,SanDiego,California,92121 AlanL.Lehman(17), DivisionofHema-
tology and Oncology, Department of In-
ChristopherP.Holmes(9),AffymaxInc., ternal Medicine, UC Davis Cancer
4001 Miranda Avenue, Palo Alto, Center, University of California Davis,
California,94304 Sacramento,California,95817
contributors to volume369 xi
RuiwuLiu(15,17), DivisionofHematol- E.R. Palmacci (13), 77 Massachusetts
ogy and Oncology, Department of In- Avenue,T18-209,Cambridge,Massachu-
ternal Medicine, UC Davis Cancer setts,02139
Center, University of California Davis,
Sacramento,California,95817 Yijun Pan (9), Affymax Inc., 4001 Mi-
randa Avenue, Palo Alto, California,
MatthiasLormann(7),Kekule´-Institutfu¨r 94304
Organische Chemie und Biochemie der
Rheinischen, Friedrich Wilhelms Univer- JackG.Parsons(3),MimotopesPtyLtd.,
sita¨t Bonn, Gerhard-Domagk-Strasse 1, 11 Duerdin Street, Clayton, Victoria,
Bonn,D-53121,Germany 3168,Australia
Jan Marik (15), Division of Hematology Robert Pascal (10), UMR 5073, Univer-
and Oncology, Department of Internal site´ de Montpellier 2, CC017, place
Medicine,UCDavisCancerCenter,Uni- Euge`ne Bataillon, Montpellier Cedex 05,
versity of California Davis, Sacramento, F-34094,France
California,95817
Clemencia Pinilla (18), Torrey Pines In-
Katia Martina (23), Discovery Research stituteforMolecularStudiesandMixture
Oncology, Pharmacia Italy S.p.A., Viale
Sciences, Inc., 3550 General Atomics
Pasteur10,Nerviano(MI),I-20014,Italy
Court,SanDiego,California,92121
JoesephMaxwell(17),DivisionofHema- Obadiah J. Plante (13), Massachusetts
tology and Oncology, Department of In-
Institute of Technology, Department of
ternal Medicine, UC Davis Cancer
Chemistry, 77 Massachusetts Avenue,
Center, University of California Davis,
Cambridge,Massachusetts,02139-4307
Sacramento,California,95817
Gregory Qushair (2), University
WimMeutermans(14),AlchemiaPtyLtd.,
of Barcelona, Barcelona Biomedical
3 Hi-Tech Court, Brisbane Technology
Research Institute, Barcelona Science
Park,EightMilePlains,QLD4113,Aus-
Park, Josep Samitier 1, Barcelona,
tralia
08028,Spain
George C. Morton (4), Rhoˆne-Poulenc
JorgRademann(21),Eberhard-Karls-Uni-
Rorer, 500 Arcola Road, Collegeville,
versity, Tu¨bingen, Institute of Organic
Pennsylvania,19426
Chemistry,AufderMorgenstelle18,Tu¨-
AdelNefzi(25),TorreyPinesInstitutefor bingen,72076,Germany
MolecularStudies,3550GeneralAtomics
Court,SanDiego,California,92121 Joseph M. Salvino (8), Director of Com-
binational Chemistry, Adolor Corpor-
Thomas Nixey (24), Amgen Inc., Depart- ation, 700 Pennsylvania Drive, Exton,
mentofSmallMoleculeDrugDiscovery, Pennsylvania,19345
OneAmgenCenterDrive,29-1-B,Thou-
sandOaks,California,91320 Peter H. Seeberger (13), Laboratorium
fuer Organische Chemie, HCI F 315,
JohnM.Ostresh(25),TorreyPinesInsti- Wolfgang-Pauli-Str.10,ETH-Hoengger-
tute,Room2-136,3550GeneralAtomics berg,CH-8093Zu¨rich,Switzerland
Court,SanDiego,California92121
Craig S. Sheehan (3), Mimotopes Pty
VitecekPadeˇra(6),Torvic,3251WLam- Ltd., 11 Duerdin Street, Clayton, Vic-
bertLane,Tucson,Arizona,84742 toria,3168,Australia
xii contributors to volume 369
AdrianL.Smith(24),AmgenInc.,Depart- Jesus Vazquez (2), University of Barce-
mentofSmallMoleculeDrugDiscovery, lona, Barcelona Biomedical Research
One Amgen Center Drive, Thousand Institute, Barcelona Science Park, Josep
Oaks,California,91320 Samitier1,Barcelona,08028,Spain
Re´gine Sola (10), UMR 5076, Ecole
MichaelL.West(14),AlchemiaPtyLtd.,
Nationale Supe´rieure de Chimie de
Eight Mile Plains, Queensland 4113,
Montpellier, 8, rue Delaware l’Ecole
Australia
Normale, Montpellier Cedex 05, F-
34296,France Zemin Wu (3), Mimotopes Pty Ltd., 11
AiminSong(17),UniversityofCalifornia, Duerdin Street, Clayton, Victoria, 3168,
UC Davis Cancer Center, Division of Australia
Hematology and Oncology, 4501 X
Street,Sacramento,California,95817 BingYan(1),ChemRxDivision,Discovery
PartnersInternational,385OysterPoint,
Alexander Stadler (11), Institute of
Boulevard,Suite1,SouthSanFrancisco,
Chemistry, Karl-Franzens-University
California,94080
Graz, Heinrichstrasse 28, Graz, A-8010,
Austria YongpingYu(25),TorreyPinesInstitute,
Paul Tempest (24), Amgen Inc., Depart- Room 2-136, 3550 General Atomics
mentofSmallMoleculeDrugDiscovery, Court,SanDiego,California,92121
OneAmgenCenterDrive,29-1-B,Thou-
sandOaks,California,91320 Florencio Zaragoza (26), Medicinal
Chemistry,NovoNordiskA/S,NovoNor-
David Tumelty (9), Affymax, Inc.,
diskPark,Malov,2760,Denmark
4001 Miranda Avenue, Palo Alto,
California,94304 JiangZhao(1),ChemRxDivision,Discov-
Josef Vagner (16), Department of Chem- ery Partners International, 385 Oyster
istry,UniversityofArizona,Tuscon,Ari- Point Boulevard, Suite 1, South San
zona,85741 Francisco,California,94080
[1] high-throughput LC/UV/MSanalysis of libraries 3
[1] High-Throughput Parallel LC/UV/MS Analysis of
Combinatorial Libraries
By Liling Fang, Jiang Zhao, and Bing Yan
Introduction
Combinatorial chemistry and high-throughput organic synthesis allow
thepreparationofalargenumberofdiversecompoundsinarelativeshort
period of time in order to accelerate discovery efforts in the pharmaceut-
ical and other industries. A library can comprise hundreds to thousands
ofcompounds with theneedtorapidlyanalyzethose compoundsfortheir
identityandpurity.Differentcompoundseparationandmassspectrometry
(MS)techniqueshavebeenappliedforthecharacterizationofcombinator-
iallibraries.Theseincludeseparationtechniquessuchasliquidchromatog-
raphy(LC)andcapillaryelectrophoresisanddifferent ionizationmethods
andmassanalyzers.1–3LC/MS*isthemostpopulartechniqueusedincom-
binatorial library analysis because it combines separation, molecular
weight determination,andrelativepurityevaluation inasinglesamplein-
jection. However, the throughput of conventional LC/MS could not meet
the need to analyze every member in a large combinatorial library in a
timely fashion.
Higher-throughput analysis was achieved by utilizing shorter columns
at higher flow rates.4 Supercritical fluid chromatography (SFC)/MS has
1A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’
(M.E.Swartz,ed.).MarcelDekker,NewYork,2000.
2B.Yan,‘‘AnalyticalMethodsinCombinatorialChemistry.’’Technomic,Lancaster,2000.
3D.G.Schmid,P.Grosche,H.Bandel,andG.Jung,Biotechnol.Bioeng.Comb.Chem.71,
149(2001).
*Abbreviations:CLND,chemiluminescencenitrogendetection;ClogP,calculatedpartition
coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization
mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC,
liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chroma-
tography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry –
mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV
detector; LIB, compound library; log P, water/octanol partition coefficient; MUX,
multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography;
TFA,trifluoroaceticacid;TIC,totalioncurrent;TOF,timeofflight;TOFMS,timeofflight
massspectrometry.
4H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass
SpectrometryandAlliedTopics,Dallas,Texas,June13–17,1999.
Copyright2003,ElsevierInc.
Allrightsreserved.
METHODSINENZYMOLOGY,VOL.369 0076-6879/03$35.00
4 analytical techniques [1]
beenusedtoachievedesirablehighspeedtakingadvantageofthelowvis-
cosityofCO .5However,theserialLC/MSapproachbyitsnaturedoesnot
2
match the speed of parallel synthesis. Parallel LC/MS is the method of
choiceto increasethroughputwhilemaintainingthe separationefficiency.
An eight-probe Gilson 215/889 autosampler was incorporated into a
quadruple mass spectrometer.6 This arrangement enabled the injection of
eight samples (a column from a 96-well microtiter plate) simultaneously
for flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput
of 8 samples/min. A novel multiplexed electrospray interface (MUX)7
was developed in 1999 and became commercially available for parallel
high-throughput LC/UV/MS analysis. The eight-way MUX consists of
eight nebulization-assisted electrospray ionization sprayers, a desolvation
gas heater probe, and a rotating aperture. It can accommodate all eight
high-performanceliquidchromatograph(HPLC)streamsatareducedflow
rateof<100 (cid:2)l/minperstreamandconductelectrosprayionizationforall
eight streams simultaneously. Ions are continuously formed at the tip of
eachsprayerandtheMUXinterfaceallowssprayerstobesampledsequen-
tiallyusingtherotatingaperturedrivenbyaprogrammablesteppermotor.
At any given time, only ions from one stream are admitted into the ion
samplingcone,whileionsfromtheothersevensprayersareshielded.Each
liquidstreamissampledforapresettimewithmassspectraacquiredinfull
massrangeintoeightsimultaneouslyopendatafilessynchronizedwiththe
spray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-s
intersprayerdelaytime,thetime-of-flight(TOF)massanalyzercanacquire
a discrete data file of electrospray ion current sampled from each stream
over the entire HPLC separation with a cycle time of 1.2 s. Therefore,
this eight-way MUX-LCT was like having eight individual electrospray
ionization (ESI)-MS systems working simultaneously.
The MUX interfaceenables the coupling of parallel liquidchromatog-
raphy to a single mass spectrometer. This technology has had a great
impact in high-throughput LC/MS analysis. In drug development, a four-
way MUX interface was used on a triple quadrupole mass spectrometer
to simultaneously validate LC/MS/MS methods for the quantitation of
loratadine and its metabolite in four different biological matrixes8 and of
5M.C.Ventura,W.P.Farrell,C.M.Aurigemma,andM.J.Greig,Anal.Chem.71,2410
(1999).
6T.Wang,L.Zeng,T.Strader,L.Burton,andD.B.Kassel,RapidCommun.MassSpectrom.
12,1123(1998).
7V.DeBiasil,N.Haskins,A.Organ,R.Bateman,K.Giles,andS.Jarvis,RapidCommun.
MassSpectrom.13,1165(1999).
8M.K.Bayliss,D.Little,D.M.Mallett,andR.S.Plumb,RapidCommun.MassSpectrom.
14,2039(2000).
[1] high-throughput LC/UV/MSanalysis of libraries 5
diazepaminratlivermicrosomesforinvitrometabolicstability.9Thefour-
channel LC/MS/MS system was also reported for the quantification of a
druginplasmaonboththenarrow-boreandcapillaryscales.10Byincorpor-
ating divert valves into this system, aliquots of plasma could be directly
analyzedwithoutsamplepreparation.Thefour-channelLC/MS/MShasre-
ducedmethodvalidationtime,increasedsamplethroughputby4-fold,and
afforded adequate sensitivity, precision, and negligible intersprayer cross-
talk.8,9 In protein analysis, an eight-way MUX coupled with a TOFMS
analyzer has proved to be a powerful tool to monitor the protein purifica-
tionprocessbyscreeningfractionsfrompreparativeion-exchangechroma-
tography with a throughput of 50 protein-containing fractions in less than
anhour.11
Ahigh-pressuregradientparallelpumpingsystem(JASCOPAR-1500)
hasbeendevelopedtoconducthigh-throughputparallelliquidchromatog-
raphy.12 It is a 10-pump system where two pumps are used to generate a
binary gradient and eight pumps to deliver the mixed solvent to eight LC
columns.Comparingthissystemtoaconventionalsystemwithtwopumps
orabinarypumpforLCgradientandasimplesplittertodividethegradi-
enttoeightLCcolumns,thissystemcanensureuniformflowratesthrough
eachLCcolumn.Thissystemhasbeenusedforpeptidesandcombinatorial
sample,12 protein analysis,13 and bioanalysis.9
We have optimized an eight-way MUX coupled to a TOFMS analyzer
to carry out eight-channel parallel LC/UV/MS analysis of combinatorial
libraries14 in the past 2 years. This system has not only provided the
capacity needed for library analysis, but also enabled simultaneous evalu-
ation of experimental parameters to expedite the method development
process. In this chapter, we discuss the optimization of this system and
present a high-throughput protocol for combinatorial library analysis. We
also compare the eight-channel parallel LC/UV/MS system to a conven-
tional single channel LC/UV/MS system in terms of performance and
operation.
9D.Morrison,A.E.Davis,andA.P.Watt,Anal.Chem.74,1896(2002).
10L.Yang,T.D.Mann,D.Little,N.Wu,R.P.Clement,andP.J.Rudewicz,Anal.Chem.73,
1740(2001).
11B.Feng,A.Patel,P.M.Keller,andJ.R.Slemmon,RapidCommun.MassSpectrom.15,
821(2001).
12D.Tolson,A.Organ,andA.Shah,RapidCommun.MassSpectrom.15,1244(2001).
13B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73,
5691(2001).
14J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS
ConferenceonMassSpectrometryandAlliedTopics,Chicago,IL,May,27–31,2001.