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CONTRIBUTORS
KhaledAlawam
ForensicMedicineDepartment,MinistryofInterior,KuwaitCity,Kuwait
HosseinBaharvand
DepartmentofDevelopmentalBiology,UniversityofScienceandCulture,andDepartment
ofStemCellsandDevelopmentalBiologyatCellScienceResearchCenter,RoyanInstitute
forStemCellBiologyandTechnology,ACECR,Tehran,Iran
A.ElizabethBond
InstituteofMassSpectrometry,CollegeofMedicine,SwanseaUniversity,Swansea,United
Kingdom
ChristophH.Borchers
UniversityofVictoria—GenomeBritishColumbiaProteomicsCentre,andDepartmentof
BiochemistryandMicrobiology,UniversityofVictoria,PetchBuildingRoom207,Victoria,
BritishColumbia,Canada
NicolaLuigiBragazzi
NanobiotechnologyandBiophysicsLaboratories(NBL),DepartmentofExperimental
Medicine(DIMES);NanoworldInstituteFondazioneELBANicolini(FEN),Pradalunga,
Bergamo,andSchoolofPublicHealth,DepartmentofHealthSciences(DISSAL),
UniversityofGenoa,Genoa,Italy
JuanCasado-Vela
CentroNacionaldeBiotecnolog´ıa,SpanishNationalResearchCouncil(CSIC),Madrid,
Spain
EdDudley
InstituteofMassSpectrometry,CollegeofMedicine,SwanseaUniversity,Swansea,United
Kingdom
OctavioLuizFranco
CentrodeAna´lisesProteoˆmicaseBioqu´ımicas,ProgramadePo´s-Graduac¸a˜oemCieˆncias
GenoˆmicaseBiotecnologia,UniversidadeCato´licadeBras´ılia,Bras´ılia,Brazil
Jose´ ManuelFranco-Zorrilla
CentroNacionaldeBiotecnolog´ıa,SpanishNationalResearchCouncil(CSIC),Madrid,
Spain
DustinC.Frost
SchoolofPharmacy,UniversityofWisconsin,Madison,Wisconsin,USA
ManuelFuentes
CentrodeInvestigacio´ndelCa´ncer/IBMCC(USAL/CSIC),IBSAL,Departamentode
Medicina,UnidaddeProteomics&ServicioGeneraldeCitometr´ıa,Universityof
Salamanca,Salamanca,Spain
ix
x Contributors
LingjunLi
SchoolofPharmacy,andDepartmentofChemistry,UniversityofWisconsin,Madison,
Wisconsin,USA
ClaudioNicolini
NanobiotechnologyandBiophysicsLaboratories(NBL),DepartmentofExperimental
Medicine(DIMES),UniversityofGenoa,Genoa;NanoworldInstituteFondazioneELBA
Nicolini(FEN),Pradalunga,Bergamo,Italy,andBiodesignInstitute,ArizonaState
University,Tempe,Arizona,USA
EugeniaPechkova
NanobiotechnologyandBiophysicsLaboratories(NBL),DepartmentofExperimental
Medicine(DIMES),UniversityofGenoa,Genoa,andNanoworldInstituteFondazione
ELBANicolini(FEN),Pradalunga,Bergamo,Italy
BernardoA.Petriz
CentrodeAna´lisesProteoˆmicaseBioqu´ımicas,ProgramadePo´s-Graduac¸a˜oemCieˆncias
GenoˆmicaseBiotecnologia,UniversidadeCato´licadeBras´ılia,Bras´ılia,Brazil
EvgeniyV.Petrotchenko
UniversityofVictoria—GenomeBritishColumbiaProteomicsCentre,Victoria,British
Columbia,Canada
GhasemHosseiniSalekdeh
DepartmentofMolecularSystemsBiologyatCellScienceResearchCenter,RoyanInstitute
forStemCellBiologyandTechnology,ACECR,Tehran,andDepartmentofSystems
Biology,AgriculturalBiotechnologyResearchInstituteofIran,Karaj,Iran
FaezehShekari
DepartmentofMolecularSystemsBiologyatCellScienceResearchCenter,RoyanInstitute
forStemCellBiologyandTechnology,andDepartmentofDevelopmentalBiology,
UniversityofScienceandCulture,ACECR,Tehran,Iran
PREFACE
Inthelastdecade,proteomicsemergedasaveryvaluabletoolinbiomedical
and pharmacological research. Different proteomic techniques have been
employedinthescreeningforbiomarkersfordifferentdisordersanddiseases,
inunderstandingmolecularmechanismsunderlyingpathologicalalterations
in humans, in studying protein structures, design of potential therapeutics,
etc. Considering the wide application of proteomics in biomedicine and
pharmacology andtheincreasingnumberofspecialists from differentfields
employingproteomictechniques,wefocusedthisvolumeoftheAdvancesin
Protein Chemistry and Structural Biology on Proteomics in Biomedicine and
Pharmacology.
Chapter 1 in this volume presents the main classical gel-based methods
andtheadvancesofgel-freequantitativeproteomictechniques.Theappli-
cation of these proteomic methods in elucidation of host–bacteria interac-
tionsanddesignoftreatmentforanumberofinfectiousdiseasesisreviewed.
Protein phosphorylation and glycosylation play fundamental roles in
manybiologicalprocessesasoneofthemostcommon,andthemost com-
plex, posttranslational modification. Alterations in these posttranslational
modifications are now known to be associated with many diseases. As a
result, the discovery and detailed characterization of phosphoprotein and
glycoproteindiseasebiomarkersisaprimaryinterestofbiomedicalresearch.
TherehavebeenmanyadvancesinthisareaandthesearedetailedinChap-
ters 2 and 3, both in relation to available protocols for phospho/glyco-
proteomic analysis and to the widening range of biomedical fields in
which such approaches are being commonly applied. A special emphasis
is given to their application to cancer biomarkers and neurodegenerative
diseases.
Nextfivechaptersreviewindetailstheuseofdifferentproteomictech-
niques in studying oraldiseases (Chapter4), alterations in protein structure
anddesignofpersonalizedtreatment(Chapters5and6),stemcellsorganelle
proteomics research and challenges in subcellular proteomics (Chapter 7),
and screening of protein–protein and protein–DNA interactions and its
application in biomedicine (Chapter 8).
Thefinalchapter(Chapter9)inthisvolumefocusesontheapplicationof
differentproteomictechniquesindiagnosisandtreatmentofpsychiatricdis-
orders such as major depression, suicidal behavior, schizophrenia, and
xi
xii Preface
attentiondeficit/hyperactivitydisorder.Thepotentialofspecificbiomarkers
determined by proteomic tools for distinguishing between comorbid psy-
chiatricdisordersinclinicalsetupaswellastheirpotentialforunderstanding
mechanisms underlying the disorders and in discovery of new treatment
strategies is also discussed.
Theaimofthisvolumeistopromotefurtherproteomic-basedstudiesin
biomedicine and pharmacology in order to discover reliable tools for early
diagnosis and treatment/management of different diseases and disorders.
ROSSEN DONEV
Singleton Park
Swansea University
Swansea, UK
CHAPTER ONE
Application of Cutting-Edge
Proteomics Technologies for
–
Elucidating Host Bacteria
Interactions
Bernardo A. Petriz, Octavio Luiz Franco1
CentrodeAna´lisesProteoˆmicaseBioqu´ımicas,ProgramadePo´s-Graduac¸a˜oemCieˆnciasGenoˆmicase
Biotecnologia,UniversidadeCato´licadeBras´ılia,Bras´ılia,Brazil
1Correspondingauthor:e-mailaddress:[email protected];[email protected]
Contents
1. Introduction 2
2. ClassicalProteomicsStrategiesforBiomedicalResearchinGeneral 2
2.1 Gel-basedmethods 4
3. Gel-FreeMethods 5
3.1 Gel-free-labelingmethods 6
3.2 Label-freeandabsolutequantification 8
4. NewProteomicMethodsinLookingforBacterialPathogens 9
5. ProteomicAdvancesinLookingforHostOrganisms 13
6. Prospects 17
References 18
Abstract
Advanced techniques and high-throughput protein analysis have led proteomics to
substantive progress in the understanding of bacterial–host interactions. Mass spec-
trometry (MS)-based proteomics have been a central methodology in the discovery
of new protein involved in the infectious process that leads to thousands of deaths
everyyear.Thediscoveryofnovelproteintargets,togetherwithdenovodrugdesign,
improves the accuracy of early diagnosis, leading to improved new treatments.
MS-basedproteomicshasalsobeenwidelyappliedtostructuralbiology,whereprote-
omicinvestigationisbeingusedtoimproveknowledgeontherelationshipbetween
proteinsequence,structure,andfunction.Thus,thesearchfortherapeutictargetsfor
infectiousdiseasesusingthesecutting-edgetechnologiesrepresentsthenewfrontiers
forproteomicsapplicationsinbiomedicineandpharmacology.Inthisreview,themain
classicalgel-basedmethods(2-DE,DIGE)arediscussed,aswellastheadvancesofgel-
freequantitativeproteomictechniques,frommetabolicandchemicallabeling(SILAC,
iTRAQ,ICAT,16O/18O,QconCAT)tononlabeling(MSspectracountingandpeakintegra-
tion) strategies. Together, these proteomic methods are currently being used in the
AdvancesinProteinChemistryandStructuralBiology,Volume95 #2014ElsevierInc. 1
ISSN1876-1623 Allrightsreserved.
http://dx.doi.org/10.1016/B978-0-12-800453-1.00001-4
2 BernardoA.PetrizandOctavioLuizFranco
quest for tailor-made pharmaceutical and biomedical research for bacterial control,
whereadvancesintheseanalyticalmethodsmayrepresentgreaterimprovementsin
thetreatmentofanumberofinfectiousdiseases.
1. INTRODUCTION
In recent decades,infectious diseasescaused by microorganisms have
becomeanincreasinghealthproblem.Bacterialinfectionscausedbyresistant
strains are of graveconcern in numerous hospitals around the world, espe-
cially in elderly patients, those compromised by illness and those receiving
immunity-suppressantdrugs(Grundmannetal.,2011).Inthiscontext,itis
essential to improve the understanding of infectious mechanisms and the
hostresponseinordertodevelopdrugswithpotentialactivityagainstthese
pathogens.Tofillthemanifoldgapsthatremaininourunderstandingofbac-
terial infectious processes, proteomics has been widely used (Cox et al.,
2012). In recent years, proteomic tools have accomplished significant
advances in the characterization of proteins involved in the mechanism of
infectious pathogens and also in the patient’s response (Lima et al., 2013).
In this context, this review focuses on proteomics tools used in the better
understandingofproteinsinvolvedininfectiousprocessesinmicroorganism
andmammals,providingabroadoverviewofproteinspossiblyrelatedtothe
resistance process.
2. CLASSICAL PROTEOMICS STRATEGIES
FOR BIOMEDICAL RESEARCH IN GENERAL
Theprominentroleofproteinsinallbiologicalprocess,herewithspe-
cial attention to pathogenesis and pathophysiology, has made the study of
proteinsbecomewidelyincorporatedintoanumberoffieldsinbiomedical
research, which include biomarker discovery and novel drug design (De
Masi, Pasca, Scarpello, Idolo, & De Donno, 2013; Oswald, Groer,
Drozdzik,&Siegmund,2013;Parguina,Rosa,&Garcia,2012).Inthiscon-
text, the discovery of protein targets associated with infectious pathologic
developmentrepresentsanadvanceinearlydiagnosisanddrugdevelopment
(Bougnoux & Solassol, 2013; Ghafourian, Sekawi, Raftari, & Ali, 2013;
Konvalinka, Scholey, & Diamandis, 2012; Oswald et al., 2013). In recent
years, this objective has produced a substantial amount of proteomic data,
especially associated with phenotypes derived from abnormal protein
Cutting-EdgeProteomicsTechnologiesforElucidatingHost–BacteriaInteractions 3
profiling and biomarker discovery (Banks et al., 2000; Castagna, Polati,
Bossi, & Girelli, 2012).
Proteomicsisanensembleoftoolsusedtorevealastaticprofileofpro-
teins expressed in a complex system resulting from dynamic biological sig-
nalingandgeneregulation.Inthisway,proteomicanalysisaimstoidentify
andverifytheroleofagivenproteinormoreprecisely,acollectionofgene
products in biological processes (e.g., in pathology) (Domon & Aebersold,
2006).Thisprocessisoftenchallenging,duetothecomplexdynamicrange
and heterogeneity of several proteome samples (e.g., plasma and tissue if
much greater than genome size) (Anderson & Anderson, 2002; Harrison,
Kumar, Lang, Snyder, & Gerstein, 2002). Moreover, proteomic analysis
mayalsobelaboriousandtime-consuming,sometimesinvolvingseveralsets
of biologic and technical analysis to overcome possible failures in technical
reproducibility. Despite these challenges, as a great advance, some proteo-
mic analysis may resolve thousands of proteins/peptides simultaneously
(Tang, Beer, & Speicher, 2011).
Proteomic techniques may be divided into gel-based and gel-free
methods, but this division does not limit the interaction of both method-
ologies, frequently seen in several studies as complementary strategies
(Charro et al., 2011; Jungblut et al., 2010; Selvaraju & El Rassi, 2011;
Thierolf et al., 2008). Gel-based techniques are represented mainly by
the classic 1D, 2-DE, and 2D-DIGE, the ultimate evolution of classic
two-dimensionalelectrophoresis(Minden,2012).Otherwise,gel-freeana-
lyses are conducted by a wide range of liquid chromatography strategies
(e.g., HPLC, UPLC, nanoLC, MudPIT) which are often directly coupled
to automated mass spectrometry (MS) apparatus (e.g., LC/MS) (Franzel &
Wolters, 2011; Mitulovic & Mechtler, 2006; Nagele, Vollmer, Horth, &
Vad, 2004). In addition, protein/peptides may be labeled in advance to
LC/MS for absolute and/or relative proteome quantitation, enhancing
quantitative proteomic analysis (May et al., 2011). Independently of the
chosenstrategy,MSanalysisisacentraltechnologyandkeystepforsimple
andhigh-throughputproteomiccharacterizationandanalysis(protein/pep-
tide identification) (Domon & Aebersold, 2006; May et al., 2011). More-
over, MS is fundamental for identifying posttranslational modifications
(PTMs), a key molecular signaling process, highly investigated by
MS-based proteomics (Cravatt, Simon, & Yates, 2007; Zhao & Jensen,
2009), since some PTMs such as phosphorylation are associated with the
development of clinical conditions (e.g., Alzheimer’s, cardiovascular dis-
ease, cancer) (Kolarova, Garcia-Sierra, Bartos, Ricny, & Ripova, 2012;
4 BernardoA.PetrizandOctavioLuizFranco
Thakur et al., 2008; Toepfer et al., 2013; Trombino et al., 2004; Walker,
Fullerton, & Buttrick, 2013).
Hence, biomedical and pharmacology fields have benefited from the
great advances in MS-based proteomics, fundamental for high-throughput
biomarker screening and development of novel pharmacologic strategies
(Berna et al., 2008; Thierolf et al., 2008; Vasudev et al., 2008; Yang
et al., 2011). In this review, the application of MS-based proteomics tools
and strategies in biomedical and pharmacologic fields will be addressed
for bacterial control and the treatment of a number of infectious diseases.
Section1focusesontheproteomictoolsusedforquantitativeandqualitative
analysis followed by their application in the research of host–bacterial
interactions.
2.1. Gel-based methods
Two-dimensional gel electrophoresis (2-DE) is still the most widely used
method in quantitative and qualitative proteomic studies and is the only
technique that can resolve up to 10,000 protein species from large sets of
complex protein mixtures (May et al., 2011; Wittmann-Liebold,
Graack,&Pohl,2006).Thistechnologyseparatesthesamplesbytwocon-
secutivetechniques:isoelectricfocusing,whichdiscriminatesproteinsbased
on their isoelectric point, followed by sodium dodecyl sulfate polyacryl-
amidegelelectrophoresis(SDSPAGE),whichdiscriminatesproteinsbased
on their molecular weight (Gorg, Weiss, & Dunn, 2004).
Despite the amplitude of 2-DE application, the technique is extremely
laborious,time-consuming,andmoresensitivetotechnicalreproducibility
error,sincelargesetsofgelrepetitionandsampleareusuallyneeded(Petriz,
Gomes,Rocha,Rezende,&Franco,2012).Thus,limitedsampleavailabil-
ity is an issue in 2-DE analysis, especially concerning poor protein extrac-
tion. Moreover, 2-DE technique also fails to resolve low abundant and
hydrophobic proteins as well as those with molecular size out of the range
of5–150kDaorwithextremepHrange(<3.5and>10)(Mayetal.,2011).
Themajorityoftheselimitationswereovercomebythedevelopmentofdif-
ferential gel electrophoresis (DIGE) (Unlu, Morgan, & Minden, 1997).
DIGE is the ultimate evolution of 2-DE technique, which significantly
improved the analytical power of gel-based methods in proteome research
(Minden,2012).Theseimprovementsarebasedonsignificantlyenhancing
technical reproducibility and quantification over different proteome sam-
ples, previously labeled with spectrally resolvable fluorophore agents
Cutting-EdgeProteomicsTechnologiesforElucidatingHost–BacteriaInteractions 5
(CyDyes™: Cy2, Cy3, and Cy5; GE Healthcare Europe GmbH). After
labeling, samples are pooled together with an internal standard consisting
ofthemixtureofallsamples,whichleadstoamoreaccuratenormalization
of protein spots from all sample gels. When the gel is digitalized using dif-
ferent wavelengths, each particular fluorophore is excited, generating dis-
tinct gel images corresponding to each prelabeled proteome sample. The
abundance of each protein spot is measured as a ratio to its corresponding
spotpresentintheinternalstandard,bysoftwareprograms,andthestatisti-
callysignificantchangesinspotsaremarkedforfurtherMSanalysis(Scherp,
Ku, Coleman, & Kheterpal, 2011). In this way, DIGE followed by MS is
extremely useful for characterizing differential proteome expression
(Winnik et al., 2012). This gel-based technique may also be used together
withgel-freemethods(e.g.,LC/MS,LC-MS/MS),improvingitsanalytical
power (Frohlich et al., 2006; Lee et al., 2012; Raggiaschi et al., 2006;
Weeks, 2010).
3. GEL-FREE METHODS
Asmentionedbefore,alimitedsampleisacommonsituationinsev-
eralbiomedicalfields(e.g.,rarecancerandinvasiveprocedures),sometimes
becomingarestrictiveissueforsomeproteomictechniquessuchashugesets
of2-DEgels,whichusuallyrequireahighamountofsample.Therefore,the
option for gel-free methods is often applied as an alternative to gel-based
techniques, since a low amount of sample is required and because peptide
mixture is less complex to analyze compared to proteins (May et al.,
2011). The direct connection of LC to MS analyzers leads these methods
to be referred to as MS-based methods.
AtypicalLC-MSworkflowbeginswiththeproteinsamplebeingenzy-
matically digested (e.g., trypsin, chymotrypsin, Lys-C, Glu-C, Asp-N),
with the resultant peptide mixture being separated by 1D or multi-
dimensional chromatography (HPLC or UPLC, also performed in nano-
scale) depending on its complexity. Eluted peptides are then loaded
directly (on line) to electrospray ionization (ESI) for further MS analysis
(e.g., LC-ESI-MS, LC-ESI-MS/MS). After LC separation, digested pep-
tidesmayalsobeconnectedindirectly(offline)toMSbyautomatedloading
of eluted fractions into MALDI steel plates for solid ionization process and
subsequent MS analysis (e.g., LC-MALDI-MS, LC-MALDI-MS/MS)
(Bodnar, Blackburn, Krise, & Moseley, 2003). After MS analysis, data on
peptide masses and fragmentation of ion masses are researched against a
