Proteomic Profiling and Analytical Chemistry, Second Edition: The Crossroads

279 Pages·2016·5.361 MB·English

by  Ciborowski, Paweł, Silberring, Jerzy

PROTEOMIC PROFILING
AND ANALYTICAL
CHEMISTRY
The Crossroads
SECOND EDITION
Edited by
P. CIBOROWSKI
University of Nebraska Medical Center, Omaha, NE, United States
J. SILBERRING
AGH University of Science and Technology, Krakow, Poland;
Polish Academy of Sciences, Zabrze, Poland
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LIST OF CONTRIBUTORS
J.Adamec
University of Nebraska e Lincoln, Lincoln, NE,
United States
A. Bodzo(cid:1)n-Kułakowska
AGHUniversityofScienceandTechnology,Krakow,
Poland
C.Boone
University of Nebraska e Lincoln, Lincoln, NE,
United States
A. Burns
University of Nebraska Medical Center, Omaha, NE,
United States
P.Ciborowski
University of Nebraska Medical Center, Omaha, NE,
United States
A. Drabik
AGHUniversityofScienceandTechnology,Krakow,
Poland
K.Frederick
University of Nebraska Medical Center, Omaha, NE,
United States
V.Havlí(cid:3)cek
InstituteofMicrobiology,AcademyofSciencesofthe
Czech Republic, Prague,Czech Republic
J.McMillan
University of Nebraska Medical Center, Omaha, NE,
United States
J.Meza
University of Nebraska Medical Center, Omaha, NE,
United States
xiii
xiv LIST OFCONTRIBUTORS
P.Mielczarek
AGHUniversityofScienceandTechnology,Krakow,
Poland
P.Novák
InstituteofMicrobiology,AcademyofSciencesofthe
Czech Republic, Prague, Czech Republic
P.Olszowy
University of Nebraska Medical Center, Omaha, NE,
United States; Polpharma SA Pharmaceutical Works,
(cid:1)
StarogardGdanski,Poland
F.Qiu
University of Nebraska Medical Center, Omaha, NE,
United States
J.Silberring
AGHUniversityofScienceandTechnology,Krakow,
Poland; Polish Academy of Sciences,Zabrze,Poland
M. Smoluch
AGHUniversityofScienceandTechnology,Krakow,
Poland
P.Suder
AGHUniversityofScienceandTechnology,Krakow,
Poland
J.Wiederin
University of Nebraska Medical Center, Omaha, NE,
United States
M. Wojtkiewicz
University of Nebraska Medical Center, Omaha, NE,
United States
F.Yu
University of Nebraska Medical Center, Omaha, NE,
United States
PREFACE
Theterm“proteomics”wascoinedinthemid-1990s;
however, the history of proteomics dates back to the
mid-1950sifweconsiderthefirstscientificreporton
2-dimensional electrophoresis (“Two-dimensional
electrophoresis of serum proteins.” Smithies, O. and
Poulik MD. Nature. 1956, 177(4518):1033. PMID:
13322019). Many laboratories used 1- and 2-dimen-
sional electrophoresis for protein analyses, and even
though it was not termed “profiling,” it was very
similar to what we now use in proteomic research.
More recently, soft ionization and development of
mass spectrometry sequencing of peptides and even
intact proteins, widely opened the possibilities for
globalproteinanalysis.Suddenly,wefoundourselves
in the middle of something that was growing rapidly
and extremely attractive to pursue scientifically. Our
enthusiasmforproteomicsisstillgrowingasweenter
new frontiers with the development of analytical
instrumentation (mass spectrometers, Ultra High
Pressure Liquid Chromatography, instruments for
nano-flow, analyses, etc.) and computational capa-
bilities of data analysis. We strongly believe that
aholisticapproachwillrevealmuchknowledgewhich
isyetnotknown.Wehavelearnedthatproteomicsis
a highly interdisciplinary approach but carries a risk
of false-positive results if not properly controlled at
theanalyticallevel.Hencewelearnedthatproteomics
is still short of many standards and widely accepted
quality controls. Such standards and quality control
measures will be built because of our collective
experience and to some extent based on “trial and
error” experiments. The field of proteomics is very
dynamic technologically, with new tools for sample
preparation, sample analyses and data processing
beingannouncedalmosteveryday.Toolsthatweuse
todaymightbeeasilyreplacedtomorrowbynewand
greatlyimprovedones.
It is not an easy task to prepare yet another book
on proteomics but we do hope that the content of
xv
xvi PREFACE
thissecondeditionofourbookwillstimulatereaders
and their interest in using a proteomics approach
with care, for the benefit of expansion of our knowl-
edge.Ourbookisaimedatthoseresearcherswhoare
looking for a relatively compact guide that can walk
them through major points of proteomic studies
without greatdetailfor eachandeverystepbutwith
a focus on quality control elements, frequently
overlooked during daily work maintaining basic
concepts and principles of proteomic studies.
Therefore, Proteomic Profiling and Analytical Chem-
istry: The Crossroads is written for an audience at
variouslevels,technologists/technicians,undergrad-
uate and graduate students, post-doctoral fellows,
scientists as well as principal investigators, to high-
light key points ranging from experimental design
and biology of systems in question to analytical
requirements andlimitations.
We are indebted to all of our colleagues,
coworkers, and students for their excellent contri-
butions to this book. This bookcould not have been
prepared without the extensive editorial work of
Elsevier. Thank you all for your efforts and also for
pushing us to complete materials for printing. As
always, we have to say that nobody’s perfectandwe
wouldbegratefulforanycommentsandsuggestions
thatmayleadtotheimprovementoffutureeditions.
P. Ciborowski and J. Silberring
1
INTRODUCTION
J. Silberring
AGHUniversityofScienceandTechnology,Krakow,Poland;
PolishAcademyofSciences,Zabrze,Poland
P. Ciborowski
UniversityofNebraskaMedicalCenter,Omaha,NE,United
States
CHAPTER OUTLINE
1.1 Why Do AnalyticsMatter? 1
1.2 Expectations:Whoand What? 3
1.3 What IsNext and Where Are We Going? 4
1.1 Why Do Analytics Matter?
Thesumoftheoptimalstepsintheanalyticaland
proteomic analysis (process) is not equal to the
optimal process in its entirety! As much as it is
a trivial statement, which most of us accept to be
true,ithasnotbeenfullyappreciateddespitehaving
a profound impact on the success of laborious,
expensive and, in many instances, lengthy projects,
as proteomic studies are multistep tasks involving
a variety of methods, each governed by its own
strengths and limitations. The concept of a proteo-
micstudycanbedepictedinmanyways.InFig.1.1,
weintentionallyhighlightedanalyticalcomponents/
phases because the same rules of analytical chem-
istry/biochemistry apply to discovery as well as
validation experiments. The experimental design
will be governed by a set of different rules, which
does not include instrumentation but has biology
heavily involved.
As can be observed, bioinformatics analyses are
not depicted in this model, as it is focused on
ProteomicProfilingandAnalyticalChemistry.http://dx.doi.org/10.1016/B978-0-444-63688-1.00001-X 1
(cid:1)2016ElsevierB.V.Allrightsreserved.
2 Chapter 1 INTRODUCTION
PROTEOMICS
Experimental Analytical phase I Data analysis
design phase phase
Performance of
Detection level instrumentation
Bioinformatics
Your question Reproducibility
Sample loss
New experiments
Data
New questions
Knowledge Analytical phase II Information
Validation
Figure 1.1 Schematicrepresentationofaproteomicstudy.
analytics.Bioinformaticswillbegovernedbyitsown
set of rules which are applied to the validation of
algorithms. Nevertheless, when looking at constitu-
ents of a proteomic study, we realized that the
scientist conducting such experiments must grasp
the overview of not only how biological systems
work, but also analytical boundaries for sample
preparation,fractionation,andmeasurements,tools
for database searches, statistics, and eventually
bioinformatics tools for data analysis. Because of
their complexity, proteomic studies should be con-
ductedbyateamofexperts.Astheproteomicsfield
evolves,thecollectiveexperiencefromanincreasing
number of studies inevitably leads to widely
accepted quality criteria. Although significant prog-
resshas been made,many questions about uniform
qualitycontrolcriteriaremaintobeanswered.Such
answers will result from systematic studies con-
ducted across many laboratories, platforms, and
biological systems (models). Therefore, in this book
we attempt to highlight in a short, yet comprehen-
sive manner, the impact of the basic principles of
analytical chemistry/biochemistry on the final
success of a proteomic experiment. We hope that
this point of view will help both biologists and
chemists to better understand all components of
complex proteomic study.
Chapter1 INTRODUCTION 3
1.2 Expectations: Who and What?
If two scientists, a biologist and chemist, sit at
atableanddiscussproteomicmethodology,theywill
likely emphasize different aspects of the same study,
which in each viewpoint is critical for a successful
outcome.Moreover,theyquiteoftenspeakintechnical
languagethatisnotfullyunderstoodbytheother.This
is because chemists are focused on sensitivity and
accuracyofanalyticalmeasurements,whilebiologists
pay attention to explaining biological/pathological
effectsandarelessconcernedwithexactquantitation
ofanalytes.ThisresemblesthefamouspoembyJohn
G.Saxe,“TheBlindMenandtheElephant,”inwhich
everyonetriestoidentifytheparttheyaretouching(ie,
biologist/chemist) but nobody can get a sense of the
whole system (ie, proteomic study). Biologists are
willingtoacceptahighrangeofresponses,resultingin
high standard deviations showing or indicating
“trends”indatabehaviorthatsupporttheirhypothesis.
Chemists, on the other hand, expect data to be
expressed by numerical values with high precision,
accuracy,reproducibility,andlowstandarddeviation.
Indeed, as much as precision of analytical measure-
mentsisimportant,inmanyinstances,sucheffortswill
notimprovetheoveralloutputdiscriminatingbetween
true and false. This is mostly because, very often, an
exact correlation between quantitative change and
biological effect is not defined. For example, how
importantisittomeasureadifferencebetweenlevels
ofproteinexpression above10-foldchangewhenthe
response of biological system is already saturated
bythe5-foldchangeofthisprotein?Asimilarquestion
mayarisefromenzymology,wherethemostimportant
factor is enzymatic activity and not the protein
expressionmeasuredbyatypicalproteomicapproach,
whichwillalsomeasureinactiveenzymes.Ifwebring
statisticiansandbioinformaticianstothesametableas
the biologist and chemist, which veryoften happens,
the discussion becomes even more complicated. As
illustratedinFig.1.2,ourquestioniswhatdoweseeon
theothersideofourofficewallswhenwelookforthe
expertiseofourfellowcolleagues?Itiscriticalforeach
ofus to peeroutside ofthe wallsthat confine us and
beholdtheworldofthosewhosurroundus.
4 Chapter 1 INTRODUCTION
BIOLOGISTS Biostatisticians and
Chemists and Bioinformaticians
Mass Spectrometrists
Figure1.2 Whatweseeontheothersideofthewallofourofficewhenwelookintotheoffice
spaceofourfellowcolleagueswiththeirexpertise.
1.3 What Is Next and Where Are We
Going?
Sinceproteomicsmovedfromqualitativetoquan-
titativeprofilingusingliquid-phase-basedmethodsof
sample fractionation, it fully entered the domain of
analytical chemistry. As much as it is beneficial for
proteomics to have a wide range of well-established
analytical methods, the complexity of proteomic
profiling creates multipletechnical issues. First,clas-
sical analytical chemistry focuses on high accuracy
measurementsofsingleorfewcompoundsatthesame
time.Itallowsadjustingmethods ofsampleprepara-
tion and analytical parameters with specific objec-
tive(s)scarifyingmeasurementsofothercompounds,
whicharecontaminants,ratherthananalytes.Impor-
tantly, analytical chemistry exploits specific charac-
teristics of analyzed compounds and this concept
fulfillsitspurpose.Incontrast,proteomicsattemptsto
measurehundredsandthousandsofmoleculesatthe
same time which can have a wide range of chemical
characteristics (eg, posttranslational modifications of
proteinsandpeptides)andthathaveawidedynamic
rangeofconcentrations,suchasthecircumstancewith