Methods in molecular biology vol 1598 neuroproteomics methods and protocols 2nd edition

trau-The first part of the book is entitled “current reviews of neuroproteomics approaches and applications.” This part of the book encompasses six chapters that review and highlight adv

Neuro-proteomics

Firas H Kobeissy

Stanley M Stevens, Jr Editors

Methods and Protocols

Second Edition

Methods in

Molecular Biology 1598

Series Editor

John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

Department of Cell Biology, Microbiology, & Molecular Biology,

University of South Florida, Tampa, FL, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

ISBN 978-1-4939-6950-0 ISBN 978-1-4939-6952-4 (eBook)

DOI 10.1007/978-1-4939-6952-4

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Foreword I

Major health care challenges remain to be the diagnosis and treatment of stroke and matic brain injury (TBI) An improved understanding of the neurotrauma biological attri-butes of proteins and peptides is expected to enable a better understanding of molecular changes prompted by brain conditions Such understanding will substantially improve patient care Neuroproteomics is an emerging and dynamic area of research that is deserv-edly drawing immense attention This book edited by Stevens and Kobeissy is timely and provides concise sets of articles that capture recent advancements in neuroproteomics and clinical application of this dynamic area of research to understand the molecular protein changes that are directly related to the development and progression of many central ner-vous system diseases, stroke, and traumatic brain injury The book is divided into three parts covering a wide spectrum of neuroproteomics

trau-The first part of the book is entitled “current reviews of neuroproteomics approaches and applications.” This part of the book encompasses six chapters that review and highlight advances in a wide range of research activities pertaining to neuroproteomics, including exhaustive review of the advantages and challenges associated with neuroproteomics, potential of imaging mass spectrometry to study TBI and other central nerve system condi-tions, advances in degradomics and proteomics to study TBI, systems biology approach to study PTSD, and neuroproteomics in Alzheimer’s disease An attractive feature of this book that makes it useful for new and advanced researchers is the breadth of topics covered, not only in this part but throughout the book

Another attractive feature of the book is the fact that it included chapters that reviewed the current state-of-the-art areas of research as well as chapters that discussed and described experimental methods The second part of the book is dedicated to discussing and describ-ing experimental methods of neuroproteomics This part of the book constitutes the heart

of the book and included ten chapters that describe experimental methods related to toaffinity labeling, quantitative phosphoproteomics of brain tissue, glycoprotein enrich-ment in CNS, 2-DE proteomics, neuroproteomics CSF profiling by multiplexed affinity arrays, brain proteomics by IMS of parafilm-assisted microdissection-based LC-MS/MS, SILAC of primary microglia, and TBI neuroproteomics by 2-DE and Western blotting.The comprehensiveness of the book is evident by dedicating the third part of the book

pho-to “Bioinformatics and Computational Methods.” This is an important area of research and the success of all the activities described in Parts I and II hinges on the development and implementation of bioinformatics and computational tools that are critical for the auto-mated interpretation and quantitation of the “big data” generated by neuroproteomics analytical approaches This part includes five chapters The first chapter in this part describes

an algorithm capable of degradomics prediction This chapter is aligned with the review of degradomics (Chapter 4) A systems biology and bioinformatics approach to the effect of secondhand tobacco smoke on the nitration of brain proteome is the subject of the second chapter in this part Advanced “Omic” approach to identify co-regulated clusters and tran-scription regulation network with AGCT and SHOE methods is discussed in the third chapter of this part AutoDock and AutoDock tools for protein-ligand docking and an

integration of decision tree and visual analysis to analyze intracranial pressure are the jects of the fourth and fifth chapters of this part, respectively.

sub-Stevens and Kobeissy should be commended for the fine job they have done editing this book The collection of topics and the quality of the chapters are excellent and a perfect fit for an edited book in neuroproteomics The book is timely, and the breadth and depth

of topics are outstanding This book will be an excellent resource for the new and expert researcher Students and researchers will benefit from reading the book and keeping a copy handy

spectrometry proteomics/glycoproteomics and glycomics,

Lubbock, TX, USA

Foreword II

Over the past few centuries, a number of technological advances have uncovered new zons for the scientific study of the nervous system From uncovering the electrical excit-ability of neurons and the invention of the microscope to modern imaging techniques capable of visualizing molecules in a functional brain, we have come a long way in refining our speculations about brain function Today, it is possible to correlate the molecular dynamics of neuronal circuits with the mechanisms of sensorimotor transformations in the brain and to connect them all with observable behavior

hori-With every new technique, the excitement for novelty and the promise of discovery had to

be disciplined with a word of caution: a reminder that the brain is different from other organs and studying it requires vigilance against overindulgence in interpreting results.When Dr Firas Kobaissy first mentioned to me that he was about to write the second edition of this book, I said to myself here’s a much needed revision of Neuroproteomics waiting to be written! I have known Firas for more than 5 years, through which he has been focused on the use of proteomics in the study of disease and injury, including brain injury His passion for proteomics is rivaled only by his interest in the mechanisms of brain injury

In the first edition, “Neuroproteomics” presented a number of experimental proteomic approaches to the study of the central nervous system (CNS) and its dysfunction in trauma and disease In four contiguous sections, it covered animal models used in neuroproteomics research, methods for separating and analyzing subcomponents of the neuroproteome, wide-ranging approaches for proteome characterization and quantification in the CNS, in addition to other methods to translate neuroproteomic results clinically This second edi-tion offers more updated and novel protocols that encompass both brain-wide and targeted neuroproteomic topics It includes exploration of advanced methods used for neuropro-teomics research including protein quantitation by mass spectrometry, characterization of post-translational modifications, as well as bioinformatics and computational approaches Methodology chapters follow a well-organized presentation of their respective topics, start-ing with an introduction, followed by a list of materials and reagents, step-by-step repro-ducible protocols, and instructions on troubleshooting and addressing potential pitfalls It

is a cookbook for established and new scientists looking for molecular and biochemical markers of brain function and disease

I have studied the brain and its mechanisms for nearly three decades using ology, neuroanatomy, neuropharmacology, molecular, behavioral, and imaging techniques and I have taught the same over the same period My work spanned the fields of discovery and translational sciences, with clinical applications in a couple of instances If anything, my neurotrek has taught me one important lesson about the brain: it functions more like a Jeep than a Ferrari and it constantly adapts to changing circumstances This makes the outcomes

neurophysi-of reductionist neuroscience techniques—be they physiological, cellular, molecular, or teomic—too precise and limited to the experimental question at hand, reflecting mere snapshots of the brain state at a given point in time; fleeting moments that vary with chang-ing conditions

pro-Reconstructing behavioral and cognitive states from these snapshots requires more integrated conceptual questions that put together the observations of many disciplines, and push them far beyond what a single technique can offer Along those lines, an amazing unification within the biological sciences has taken place over the past few decades and it has set the stage for addressing this challenge Genomics and proteomics have unmasked surprising similarities among proteins, their functions, and their mechanisms of action throughout the body including the nervous system This has resulted in a common concep-tual framework for all cell biology including the neuron However, the more daunting chal-lenge remains a unification between the many disciplines of biology to explain the neural basis of behavior.

This final unification requires an admission, by reductionists, of the impossibility of a bottom-up reconstruction of biological systems, and an integrationist approach that does not deny or ignore the validity and results of successful reduction

This book is a step in the right direction towards unifying cellular and molecular odologies in the study of neurons Hopefully, it will be followed by similarly successful steps towards a general biological unification

Department of Anatomy, Cell Biology

and Physiological Sciences

Faculty of Medicine

Professor and Chairman,

Interfaculty Neuroscience Graduate Program

American University of Beirut, Bliss Street,

Beirut, Lebanon

The application of proteomics to the study of the central nervous system (CNS) has greatly enhanced our understanding of fundamental neurobiological processes and has enabled the identification of proteins and pathways related to the complex molecular mechanisms underlying various diseases of the CNS This field, termed neuroproteomics, has facilitated scientific discovery through major technological and methodological advances in recent

years As part of the Methods in Molecular Biology series, this new edition will include several

exciting areas of advanced methods used for neuroproteomics research including relative and absolute protein quantitation by mass spectrometry, characterization of post- translational modifications, as well as bioinformatics and computational approaches

In the introductory part of the book (Current Reviews of Neuroproteomic Approaches and Applications), we have six timely reviews of various neuroproteomic approaches such

as neuroproteomics genesis, degradomics, proteomic analysis for the identification of fluid biomarkers, mass spectrometry-based imaging, and computational methods In addi-tion to methodology, the application of neuroproteomic approaches to understand CNS disorders such as posttraumatic stress disorder and Alzheimer’s disease is also reviewed.The second part of the book focuses on experimental methods in neuroproteomics We are excited to present updated approaches for the global-scale analysis of post-translational modification analysis These post-translational modifications include phosphorylation, gly-cosylation, as well as proteolytic cleavage In addition to post-translational modification analysis, several chapters detail procedures for quantitation of protein expression using both label-free and also novel stable isotope labeling approaches In terms of label-free quantita-tion, both mass spectrometry and multiplexed affinity arrays are described in relation to protein profiling in cerebrospinal fluid and also microvesicles and exosomes derived from neuronal cells In relation to stable isotope labeling methods in neuroproteomics, two chapters detail stable isotope labeling by amino acids in cell culture (SILAC) approaches for the analysis of primary or ex vivo microglia The SILAC chapters are focused on a single CNS cell type; however, the approach can be potentially applied to other CNS cell types after appropriate optimization Moreover, specialized method chapters are presented including proteomic approaches for identification of allosteric ligand binding sites, matrix- assisted laser desorption/ionization-based imaging, and targeted analysis of protein expres-sion in a tissue-specific approach related to neuroendocrine response

bio-In addition to experimental protocol chapters, we present five chapters in the last part

of the book that are related to bioinformatic and computational approaches in teomics These chapters include a novel degradomics prediction algorithm as well as sys-tems biology and bioinformatics approaches to characterize the global-scale effects of protein nitration and to determine transcriptional regulation networks in the context of the CNS Specialized protocols are also presented that describe methods for computational assessment of protein-ligand interactions as well as a detailed decision tree for the analysis

neuropro-of intracranial pressure

Overall, this new edition provides updated and novel protocols of neuroproteomics methods that encompass both global-scale as well as targeted and specialized topics, which

Preface

are timely additions for the molecular and phenotypic analysis of the CNS and CNS-related disorders The new compilation will be of high interest among researchers and clinical sci-entists involved in the area of biomarker research and protein biochemistry Moreover, the topics covered will be of interest to molecular biologists and biochemists who have been involved in proteomics research already or even for those new to the field.

Finally, we thank all the authors for their significant effort in writing such excellent methods and review chapters for this new edition We are also sincerely grateful to each author for their patience during the compilation and final editing of this book

There are many silent workers who deserve to be acknowledged for compiling this book Our special thanks go to the authors of the chapters who provided their top quality manu-scripts, comments, and expertise

We would like to take this opportunity to thank our colleagues at the American University of Beirut, Department of Biochemistry and Molecular Genetics, Faculty of Medicine at the American University of Beirut, Lebanon, who provided help, time, techni-cal support, and resources for completing this book We also thank our colleagues at the Byrd Alzheimer’s Institute and Department of Cell Biology, Microbiology and Molecular Biology at the University of South Florida We wish to thank Hawraa Abou Raya for her editorial support We thank Mrs Iman Karout, M.Sc., who contributed to the design of the cover art, a painting featured in the office of Professor Elie El-Chaer, at the American University of Beirut, Lebanon

Acknowledgments

Contents

Preface xi Contributors xvii

Part I Current revIews of neuroProteomICs aPProaChes

1 Neuroproteomics Studies: Challenges and Updates 3

Naify Ramadan, Hussein Ghazale, Mohammad El-Sayyad,

Mohamad El-Haress, and Firas H Kobeissy

2 Progress and Potential of Imaging Mass Spectrometry Applied

to Biomarker Discovery 21

Jusal Quanico, Julien Franck, Maxence Wisztorski, Michel Salzet,

and Isabelle Fournier

3 Biofluid Proteomics and Biomarkers in Traumatic Brain Injury 45

Safa Azar, Anwarul Hasan, Richard Younes, Farah Najdi, Lama Baki,

Hussein Ghazale, Firas H Kobeissy, Kazem Zibara, and Stefania Mondello

4 Degradomics in Neurotrauma: Profiling Traumatic Brain Injury 65

Hadi Abou-El-Hassan, Fares Sukhon, Edwyn Jeremy Assaf, Hisham Bahmad, Hussein Abou-Abbass, Hussam Jourdi, and Firas H Kobeissy

5 Evolving Relevance of Neuroproteomics in Alzheimer’s Disease 101

Simone Lista, Henrik Zetterberg, Sid E O’Bryant, Kaj Blennow,

and Harald Hampel

6 Genome to Phenome: A Systems Biology Approach to PTSD

Using an Animal Model 117

Nabarun Chakraborty, James Meyerhoff, Marti Jett,

and Rasha Hammamieh

Part II exPerImental methods

7 Photoaffinity Labeling of Pentameric Ligand-Gated Ion

Channels: A Proteomic Approach to Identify Allosteric

Modulator Binding Sites 157

Selwyn S Jayakar, Gordon Ang, David C Chiara,

and Ayman K Hamouda

8 Quantitative Phosphoproteomic Analysis of Brain Tissues 199

Bing Bai, Haiyan Tan, and Junmin Peng

9 Glycoproteins Enrichment and LC-MS/MS Glycoproteomics

in Central Nervous System Applications 213

Rui Zhu, Ehwang Song, Ahmed Hussein, Firas H Kobeissy,

and Yehia Mechref

10 A Novel 2-DE-Based Proteomic Analysis to Identify Multiple Substrates

for Specific Protease in Neuronal Cells 229

Chiho Kim and Young J Oh

11 Neuroproteomic Profiling of Cerebrospinal Fluid (CSF)

by Multiplexed Affinity Arrays 247

Anna Häggmark-Månberg, Peter Nilsson, and Jochen M Schwenk

12 Isolation and Proteomic Analysis of Microvesicles and Exosomes

from HT22 Cells and Primary Neurons 255

Richard Witas, Dale Chaput, Hirah Khan, Stanley M Stevens Jr.,

and David Kang

13 Combined MALDI Mass Spectrometry Imaging and Parafilm-Assisted

Microdissection-Based LC-MS/MS Workflows in the Study of the Brain 269

Jusal Quanico, Julien Franck, Maxence Wisztorski, Michel Salzet,

and Isabelle Fournier

14 De Novo and Uninterrupted SILAC Labeling of Primary Microglia 285

Ping Zhang, Ashley Culver-Cochran, Stanley M Stevens Jr., and Bin Liu

15 Spike-In SILAC Approach for Proteomic Analysis of Ex Vivo Microglia 295

Joao Paulo Costa Pinho, Harris Bell-Temin, Bin Liu,

and Stanley M Stevens Jr.

16 A Proteomic Evaluation of Sympathetic Activity Biomarkers

of the Hypothalamus-Pituitary-Adrenal Axis by Western Blotting

Technique Following Experimental Traumatic Brain Injury 313

Hale Zerrin Toklu, Yasemin Sakarya, and Nihal Tümer

Part III BIoInformatIC and ComPutatIonal methods

17 Efficient and Accurate Algorithm for Cleaved Fragments Prediction

(CFPA) in Protein Sequences Dataset Based on Consensus

and Its Variants: A Novel Degradomics Prediction Application 329

Atlal El-Assaad, Zaher Dawy, Georges Nemer, Hazem Hajj,

and Firas H Kobeissy

18 Effect of Second-Hand Tobacco Smoke on the Nitration

of Brain Proteins: A Systems Biology and Bioinformatics Approach 353

Firas H Kobeissy, Joy Guingab-Cagmat, Adriaan W Bruijnzeel,

Mark S Gold, and Kevin Wang

19 An Advanced Omic Approach to Identify Co-Regulated Clusters

and Transcription Regulation Network with AGCT and SHOE Methods 373

Natalia Polouliakh and Richard Nock

20 AutoDock and AutoDockTools for Protein-Ligand Docking:

Beta-Site Amyloid Precursor Protein Cleaving Enzyme 1(BACE1)

as a Case Study 391

Nehme El-Hachem, Benjamin Haibe-Kains, Athar Khalil,

Firas H Kobeissy, and Georges Nemer

21 An Integration of Decision Tree and Visual Analysis

to Analyze Intracranial Pressure 405

Soo-Yeon Ji, Kayvan Najarian, Toan Huynh, and Dong Hyun Jeong

Index 421

husseIn aBou-aBBass • Faculty of Medicine, Beirut Arab University, Beirut, Lebanon; Faculty of Medicine, Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut, Lebanon

hadI aBou-el-hassan • Faculty of Medicine, American University of Beirut Medical Center, Beirut, Lebanon

Gordon anG • Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M Health Sciences Center, Kingsville, TX, USA

edwyn Jeremy assaf • Faculty of Medicine, American University of Beirut Medical Center, Beirut, Lebanon

of Beirut, Beirut, Lebanon

hIsham Bahmad • Faculty of Medicine, Beirut Arab University, Beirut, Lebanon; Faculty

of Medicine, Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut, Lebanon

Memphis, TN, USA; Department of Developmental Neurobiology, St Jude Children’s Research Hospital, Memphis, TN, USA

Beirut, Beirut, Lebanon

harrIs Bell-temIn • Department of Cell Biology, University of Pittsburgh School

of Medicine, Pittsburgh, PA, USA

kaJ Blennow • Clinical Neurochemistry Laboratory, Department of Psychiatry

and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy

at the University of Gothenburg, Mölndal, Sweden; The Torsten Söderberg Professorship

in Medicine at the Royal Swedish Academy of Sciences, Stockholm, Sweden

adrIaan w BruIJnzeel • Department of Psychiatry and Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, USA

naBarun ChakraBorty • Integrative Systems Biology, Geneva Foundation, USACEHR, Fredrick, MD, USA

dale ChaPut • Department of Cell Biology, Microbiology and Molecular Biology,

University of South Florida, Tampa, FL, USA

ashley Culver-CoChran • Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, USA

Computer Engineering, American University of Beirut, Riad El Solh, Beirut, Lebanon

atlal el-assaad • Faculty of Engineering and Architecture, Department of Electrical and Computer Engineering, American University of Beirut, Beirut, Lebanon

nehme el-haChem • Integrative Computational Systems Biology, Institut de Recherches Cliniques de Montreal, Montreal, QC, Canada

mohamad el-haress • Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon; Faculty of Medicine, Beirut Arab University, Beirut, Lebanon

Contributors

mohammad el-sayyad • Department of Family Medicine, University of Toledo, Toledo,

University of Florida, Gainesville, FL, USA; Department of Psychiatry, School

of Medicine, Washington University, St Louis, MO, USA

Joy GuInGaB-CaGmat • Southeast Center for Integrated Metabolomics, Clinical

and Translational Science Institute, University of Florida, Gainesville, FL, USA

anna häGGmark-månBerG • Affinity Proteomics, Science for Life Laboratory, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden

BenJamIn haIBe-kaIns • Department of Medical Biophysics, University of Toronto, Toronto,

ON, Canada

Computer Engineering, American University of Beirut, Beirut, Lebanon

rasha hammamIeh • Integrative Systems Biology, US Army Center for Environmental Health Research, Frederick, MD, USA

A&M Health Sciences Center, Kingsville, TX, USA; Department of Neuroscience and Experimental Therapeutics, College of Medicine Texas A&M Health Science Center, Bryan, TX, USA; Department of Neuroscience and Experimental Therapeutics,

College of Medicine, Texas A&M Health Science Center, Kingsville, TX, USA

harald hamPel • AXA Research Fund & UPMC Chair, Paris, France; Sorbonne

Universités, Université Pierre et Marie Curie, Paris 06, Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A) & Institut du Cerveau et de la Moelle Épinière (ICM), Paris, France; Département de Neurologie, Hôpital de la Pitié-Salpêtrière, Paris, France

anwarul hasan • Department of Mechanical and Industrial Engineering, Qatar

University, Doha, Qatar; Biomedical Engineering and Department of Mechanical Engineering, American University of Beirut, Beirut, Lebanon; Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, USA; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

ahmed husseIn • Department of Biotechnology, Institute of Graduate Studies

and Research, University of Alexandria, Alexandria, Egypt

Charlotte, NC, USA

selwyn s Jayakar • Department of Neurobiology, Harvard Medical School, Boston, MA, USA

University of the District of Columbia, Washington, DC, USA

Research, Frederick, MD, USA

hussam JourdI • Faculty of Science, Department of Biology, University of Balamand, Aley, Lebanon

davId kanG • Department of Molecular Medicine, Byrd Alzheimer’s Institute, College

of Medicine, University of South Florida, Tampa, FL, USA

athar khalIl • Department of Biochemistry and Molecular Genetics, American University

of Beirut, Beirut, Lebanon

of Medicine, University of South Florida, Tampa, FL, USA

and Biotechnology, Seoul, Korea

Faculty of Medicine, American University of Beirut, Beirut, Lebanon; Department of Psychiatry, Center for Neuroproteomics and Biomarkers Research, University of Florida, Gainesville, FL, USA

sImone lIsta • AXA Research Fund & UPMC Chair, Paris, France; Sbonne Universités, Université Pierre et Marie Curie (UPMC) Paris 06, Inserm, CNRS, Institut du cerveau

et de la moelle (ICM), Département de Neurologie, Institut de la Mémoire et de la Maladie d’Alzheimer (IM2A), Hôpital Pitié-Salpêtrière, Boulevard de l’hôpital, Paris, France

of Beirut, Beirut, Lebanon

GeorGes nemer • Faculty of Medicine, Department of Biochemistry and Molecular

Genetics, American University of Beirut, Beirut, Lebanon

Peter nIlsson • Affinity Proteomics, Science for Life Laboratory, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden

rIChard noCk • Data61 & The Australian National University, Alexandria, NSW, Australia

Center, Fort Worth, TX, USA

and Biotechnology, Seoul, Korea

JunmIn PenG • Department of Structural Biology, St Jude Proteomics Facility, St Jude Children’s Research Hospital, Memphis, TN, USA; Department of Developmental

Neurobiology, St Jude Proteomics Facility, St Jude Children’s Research Hospital,

Memphis, TN, USA

Biology, University of South Florida, Tampa, FL, USA

natalIa PoloulIakh • Sony Computer Science Laboratories, Inc., Tokyo, Japan;

Department of Ophthalmology and Visual Sciences, Yokohama City University Graduate School of Medicine, Yokohama, Japan

Jusal QuanICo • Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), INSERM, U1192, Université de Lille, Lille, France

naIfy ramadan • Department of Biochemistry and Molecular Genetics, American

University of Beirut, Beirut, Lebanon

yasemIn sakarya • Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL, USA

mIChel salzet • Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse-PRISM, INSERM, U1192, Univ Lille, Lille, France

JoChen m sChwenk • Affinity Proteomics, Science for Life Laboratory, School of

Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden

ehwanG sonG • Department of Chemistry and Biochemistry, Texas Tech University,

Lubbock, TX, USA

stanley m stevens Jr • Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL, USA

fares sukhon • Faculty of Medicine, Department of Internal Medicine, American

University of Beirut Medical Center, Beirut, Lebanon

haIyan tan • St Jude Proteomics Facility, St Jude Children’s Research Hospital, Memphis,

TN, USA

hale zerrIn toklu • Department of Pharmacology and Therapeutics, University

of Florida College of Medicine, Gainesville, FL, USA; Geriatric Research Education & Clinical Center, Malcolm Randall Veterans Affairs Medical Center, Gainesville, FL, USA; North Florida Regional Medical Center, Department of Graduate Medical

Education, FL, USA

College of Medicine, Gainesville, FL, USA; Geriatric Research Education & Clinical Center, Malcolm Randall Veterans Affairs Medical Center, Gainesville, FL, USA

University of Florida, Gainesville, FL, USA; Department of Psychiatry, Center for Neuroproteomics and Biomarkers Research, Gainesville, FL, USA

maxenCe wIsztorskI • Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie

de Masse-PRISM, INSERM, U1192, Univ Lille, Lille, France

rIChard wItas • Department of Molecular Medicine, Byrd Alzheimer’s Institute, College

of Medicine, University of South Florida, Tampa, FL, USA

rIChard younes • Department of Biochemistry and Molecular Genetics, American

University of Beirut, Beirut, Lebanon

henrIk zetterBerG • Clinical Neurochemistry Laboratory, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden; Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK

Florida, Gainesville, FL, USA

TX, USA

kazem zIBara • Department of Biochemistry and Molecular Genetics, American University

of Beirut, Beirut, Lebanon

Part I Current Reviews of Neuroproteomics Approaches

and Applications

Firas H Kobeissy and Stanley M Stevens, Jr (eds.), Neuroproteomics: Methods and Protocols, Methods in Molecular Biology,

vol 1598, DOI 10.1007/978-1-4939-6952-4_1, © Springer Science+Business Media LLC 2017

Chapter 1

Neuroproteomics Studies: Challenges and Updates

Naify Ramadan, Hussein Ghazale, Mohammad El-Sayyad,

Mohamad El-Haress, and Firas H Kobeissy

Abstract

The Human Genome Project in 2003 has resulted in the complete sequence of ~99% of the human genome paving the road for the Human Proteome Project (HPP) assessing the full characterization of the translated protein map of the 20,300 protein-coding genes Consequently, the emerging of the proteomics field has successfully been adopted as the method of choice for the proteome characterization Proteomics

is a term that is used to encompass multidisciplinary approaches combining different technologies that aim

to study the entire spectrum of protein changes at a specific physiological condition Proteomics research has shown excellent outcomes in different fields, among which is neuroscience; however, the complexity

of the nervous systems necessitated the genesis of a new subdiscipline of proteomics termed as teomics.” Neuroproteomics studies involve assessing the quantitative and qualitative aspects of nervous system components encompassing global dynamic events underlying various brain-related disorders rang- ing from neuropsychiatric disorders, degenerative disorders, mental illness, and most importantly brain- specific neurotrauma-related injuries In this introductory chapter, we will provide a brief historical perspective on the field of neuroproteomics In doing so, we will highlight on the recent applications of neuroproteomics in the areas of neurotrauma, an area that has benefitted from neuroproteomics in terms

“neuropro-of biomarker research, spatiotemporal injury mechanism, and its use to translate its findings from mental settings to human translational applications Importantly, this chapter will include some recom- mendation to the general studies in the area of neuroproteomics and the need to move from this field from being a descriptive, hypothesis-free approach to being an independent mature scientific discipline.

experi-Key words Neuroproteomics, High-throughput immunoblotting, IMS, Imaging mass spectrometry

(MS), Proteomics, Human Genome Project, Human Proteome Project (HPP)

1 Introduction: Proteomics and Neuroproteomics Genesis

The completion of the Human Genome Project in 2003 has resulted in the complete sequence of ~99% of the human genome,

The global translated protein map of ~20,300 protein-coding genes

is expected to be finalized, illustrating the functional and biological characteristics of the human proteome, which will facilitate deci-phering the different role(s) of gene-coded proteins in disease and

under pathological conditions This is accomplished with the aid of proteomics and systems biology bioinformatics studies.

In the early draft of the human genome completion, around

of complexity observed in humans compared to less-developed

organisms with a relatively matched gene number (Arabidopsis thaliana has 25,500 genes) [4] This emphasized the complexity

of the proteome systems It is noted that a single gene can translate into different protein isoforms, and on average approximately ten protein isoforms/genes are translated in humans with an estimate

of one quarter of a million of different proteins and isoforms

advanced transcriptional process allowing fine and higher tion in gene expression, where it is estimated that around 3000

alternative splicing machinery driven via the genetic rearrangement

exacerbated by the presence of the different static and dynamic posttranslational modifications (PTMs) existing on different pro-

In 1994–1995, the word proteome was first coined by Marc Wilkins to denote the expression of the entire protein produced similar to the entire genes derived from the genome The pro-teome is derived from the words: PROTEin expressed by a

research in the area of proteomics where there are almost (52,525 + 10,316) articles containing the word genomics either in the abstract or in the title compared to (51,675 + 10,572) articles containing the word proteomics in the abstract or in the title (search conducted using Endnote version 17 with the PubMed database) These numbers reflect the paste at which proteomics research is progressing compared to genomics highlighting its ver-satile applications in different fields

A major characteristic of the proteome is its dynamic versatility responding to different internal and external stimuli, while the

The dynamic features of the proteome are modulated at different regulation stages of DNA transcription to mRNA, translation to polypeptides followed by the correct folding, and the insertion of the proper PTMs (glycosylation and phosphorylation) The rise in the area of proteomics was observed with the introduction of dif-ferent high-resolution approaches as advanced separation tech-niques, high-resolution mass spectrometry (MS), and versatile labeling techniques, in addition to other proteomics methods involving antibody-based approaches

Proteomics is a term that is used to encompass multidisciplinary approaches combining different technologies that aim to study the

entire spectrum of protein changes (abundance, structure, interaction, expression, and modification) at a specific physiological condition Proteomics research have shown excellent outcomes in different fields among which is neuroscience; however, the complexity of the nervous systems necessitated the genesis of a new subdiscipline of proteomics termed as “neuroproteomics.” Neuroproteomics studies involve assessing the quantitative and qualitative aspects of nervous system components encompassing global dynamic events underlying various brain-related disorders ranging from neuropsychiatric disorders (PTSD, anxiety, depression, etc.), degenerative disorders (Alzheimer’s disease, Parkinson disease, etc.), mental illness, and most importantly brain-specific neurotrauma-related injuries (traumatic brain injury

term neuroproteomics was coined for the first time in 2004 by Kim

et al.; interestingly, the authors never used “neuroproteomics” in the text which was substituted with the term “neuromics” all throughout

Neuroproteomics in conjunction with systems biology has led to revolutionize how we interpret our views on the global regulation of brain-related disorders via understanding dynamics

of protein changes (neural proteome expression, function, or

studies have been published discussing different aspects of brain- related disorders involving degenerative and neuropsychiatric dis-orders (a subdiscipline of psychoproteomics has been proposed

applica-tion on brain disorders, we will focus on brain neurotrauma as one prominent disorder that has benefited highly from the appli-cation of neuroproteomics application especially in the area of biomarker research

2 Neuroproteomics: The Study of Brain Proteome

The brain is considered among the major complex organs in the human body with a noticeable capability to perform a spectrum

of metabolic, physiological, and behavioral processes that require the intervention of several components of the nervous system at

abnormalities pertaining to this complex neural system would result in a number of brain-related disorders ranging from degenerative, neuropsychiatric, and altered mental health-related symptoms Of interest, due to the complexity of the nervous system, several advanced approaches have been developed and applied to decipher the causalities of these altered events, which focused on a number of culprits including changes in the gene/protein function/expression and interaction

On the level of the brain proteome, it is estimated that there exist around 20,000 brain proteins that are differentially expressed

chal-lenging to dissect the neuroproteome and its dynamic functions without the use of advanced separation and high-resolution pro-tein identification instrumentation Traditionally, when referring

to classical proteomics, the use of mass spectrometry coupled with advanced separation systems (online or offline with or without gel use) would be the method of choice to assess the entire spectrum

of protein characteristics This include abundance, structure, action, expression levels, and modification at a certain physiologi-cal condition [please refer to Ottens et al for detailed discussion

3 Methods in Neuroproteomics Studies

Several choices of MS-based proteomics applications have been developed; these involve the bottom-up technique vs top-down

digestion followed by MS analysis to identify peptide fragments within the complex sample mixture as applied by shotgun proteomic methods that can be applied on different biospecimens (tissue, CSF, and serum) involving nanoflow liquid chromatography (nanoLC)

pro-teomics involves the complete, intact protein analysis without the

expertise and is used for special purposes; nevertheless, the down” proteomic approach has been used to identify candidate of

above techniques can be coupled with chemical tryptic tagging with

relative and absolute quantitation (iTRAQ), stable isotope labeling with amino acids in cell culture (SILAC), or the use of super SILAC

been utilized to study neuroproteome changes as well as to assess PTM expression such as phosphorylation- dependent activitivation

where different approaches have been developed for tryptic peptide

4 Antibody-Based Neuroproteomics Approaches

Other proteomic techniques have been introduced to depict global changes that involve antibody-based techniques which are MS-free and involve a targeted detection of biomarker proteins represent-ing antigens against an antibody panel or array platforms that will

allow proteins to bind to them (Zyomyx protein biochips, BD

This approach allows a global protein discovery and has several advantages including specificity and sensitivity of the probes that can target proteins in complex high protein content milieu (CSF and serum) coupled with the rapid confirmation of the identified hits On the other hand, this technology lacks the ability to identify novel protein biomarker; in addition, it is biased toward upregu-lated protein hits Furthermore, this method lacks the ability to bind to all protein isoforms that may have different binding affinity

to the antibody arrays Finally, this technique that suffers from the probed antibodies may include low fidelity antibodies that may be

of low sensitivity to the biomarker proteins Two forms of antibody- based proteomics exist

High-throughput immunoblotting (HTPI) technology Blot™, BD Biosciences) is one novel proteomic method based on manifold immunoblotting system with usable channels that allows non-labeled samples to be PAGE resolved, and probed with mul-tiple monoclonal antibodies is HTPI which is a Western blot-based

sepa-ration data (molecular mass difference), with the advantage or requiring no bioinformatics analysis compared to regular MS data

instru-mentation compared to the MS-based techniques, and its results are easily validated since the antibody in question is already avail-able Again, the major shortcoming of this technique is the lack of exhaustiveness due to the lack of the ~30,000 different proteins and isoforms In addition, different antibody source may exhibit different affinity to define proteins as well as different species reac-tivity In our laboratory, HTPI method was used to identify a com-prehensive set of calpain and caspase-3 degradome and was

pro-teins (54 were substrates to calpain-2) (38 sensitive to caspase- 3) (48 protein were downregulated), while nine proteins were upreg-ulated post-TBI Several of the identified proteins were validated against human samples and were translated into clinical studies

Alternative to the HTPI, antibody microarray technology is designed based on DNA microarrays such as the Zymox protein

based on the concept of capturing the protein of interest using antibody-based platform By pre-labeling the protein samples from control and experimental samples using differential fluorescent Cy-3/Cy-5/Cy-2 dyes, these are probed against an antibody plat-form (standard size glass slide) leading to a differential expression

4.1 High-Throughput

Immunoblot Screening

4.2 Antibody Panel/

Microarray Approach

be achieved using additional antibodies specific to the same protein antigen, similar to the sandwich ELISA method (antibody- antigen- antibody) This method has been used to identify multiple proteins

method represents another dimension to assess known targeted protein in a high-content protein complex (CSF and serum); how-ever, similar to HTPI global approach is lacking as well

5 Neuroproteomics: Challenges

Although the brain constitutes 2% of the body mass, however, it consumes 20% of the oxygen with an abundance of 60% fat mostly localized in the myelin representing 25% of the total amount of

structures or substructures architecture with the existence of eral neural cell types including glia, astrocytes, and neurons with an approximation of 100 billion neurons and 10× more glial cells

den-drites, and forming synapses and initiating new connections

regions are small in size and are hard to obtain in sufficient amounts for analysis with the major central nervous system (CNS); proteins are either transmembrane or membrane associated (G proteins, ion

dif-ferentially in small quantities which hamper their proteomic tification due to the low copy numbers of proteins and their

sub-cellular fractionation

One major characteristic of the proteome is its dynamic tures where it reflects both temporal and spatial dynamicity depend-ing on the physiological condition as compared to the static status

fea-of the genome As discussed previously, there is a nonlinear tion between the genome and the proteome where it is challenging

rela-to draw a direct correlation and association between mRNA sion and protein translation (number of proteins from a single

alternative splicing, which is highly common in brain tissue, ating thousands of copies of highly related splices from a single gene (cadherin, e.g., has 18 different isoforms linked to morpho-

glial-spe-cific protein glial fibrillary acidic protein (GFAP) reflecting the fact

It is estimated that there exist 100% folds of complexity in the proteome compared to the genome with an average of ~10 protein

more complicated by the presence of several dynamic PTMs

reaching ~400 possible protein modifications that can render teome assessment more complicated contributing to protein com-

dynamic range of cellular proteome which reaches approximately seven orders of magnitude from one copy per cell to ten million

analy-sis of low-abundance proteins In serum, there is 0.5 pg/ml of

may exemplify the dynamic range difference of some protein

relatively straightforward task with modern MS instrumentation

espe-cially when conducting experimental work on brain tissue from animal models, the proteomics is more challenging due to the absence of amplification schemes analogous to polymerase chain reaction (PCR), and only proteins isolated from a natural source

6 Neuroproteomics Studies in Central Nervous System (CNS) Injury

Traumatic brain injury (TBI), defined as brain damage due to an external mechanical force, is among the complex neurological dis-orders that has detrimental effects on the general population Annually, it is estimated that over than two million TBI incidents occur leading to 100,000 fatalities and around 50,000 hospitaliza-

ado-lescents and adults Annually, the direct and indirect cost of TBI

complex-ity of the TBI injury events arises from the fact that it occurs in two phases mediated by different sets of proteins players activating sev-eral pro-death pathways shifting the balance from pro-survival into

events are mediated by a set of activated cysteine protease family proteins affecting several brain-specific proteins leading to an over-

primary and secondary injury phases involving different nents of the neural brain cells accompanied by dysregulation of dif-

The use of neuroproteomics applications on brain injury was aimed to understand altered protein dynamics, which benefitted mostly the field of neurotrauma especially on the levels of biomarker research Several studies have been published discussing biomarker

the fact that TBI is a complex disorder is hard to assess by current clinical techniques including the computer tomography (CT) scan and magnetic resonance imaging (MRI) which are expensive

Brain injury biomarkers represent biochemical markers that can be

of diagnostic and prognostic value capabilities that can direct brain injury treatment and management and provide insights into the

Several neurotrauma studies applying neuroproteomics

neuropro-teomics studies have assessed different modes of CNS injury involving TBI, spinal cord injury, and stroke involving experimen-tal and clinical samples and using different biofluids or brain tissue

assessed altered differential proteins in normal human postmortem immune-affinity-depleted CSF using off-gel electrophoresis, SDS-

aimed to emulate proteolytic damage in postmortem samples as observed in post-brain trauma A total of 229 proteins were identi-fied, with 172 novel protein hits that were validated by immunob-lotting and sandwich enzyme-linked immunosorbent assay (ELISA) methods In one of the TBI studies from our laboratory,

we utilized 1D difference gel electrophoresis (DIGE) to study TBI

hits were far reaching (57 downregulated and 74 upregulated in TBI), our lab utilized another advanced offline separation tech-

Another study from our laboratory designed an offline dimensional separation platform termed cation-anion exchange chromatography followed by 1D–PAGE separation (CAX-PAGE) aimed at enhancing differential comparison among samples with-out the need to mix samples with the advantage of extending the

was tested on rat cortical samples subjected to controlled cortical impact (CCI) of experimental TBI Data showed that 59 were altered (21 decreased and 38 increased) along with several novel

were validated and subjected to functional analysis

Of interest, Siman et al utilized a neuroproteomic analysis of CSF from rat model with mild/moderate TBI that was performed employing 2D–PAGE with matrix-assisted laser desorption/ion-ization time-of-flight (MALDI-TOF) MS analysis Data from this work identified several key brain injury proteins involving tau pro-

(BDP150 and SBDP120) along with several others These teins represented leaked proteins from the brain into the CSF rep-

Opii et al used a combination of 1D- and 2D–PAGE with MALDI- TOF analysis to identify the oxidized mitochondrial proteins in the rat cortex and hippocampus with experimental model of moderate

modified These proteins were involved in mitochondrial getics Several of the proteins identified were validated using

bioener-immunoblotting and immunoprecipitation techniques In an gant study by Ottens et al., post-acute brain injury urinary samples were assessed as peripheral fluids, for diagnostic urinary signature markers in brain injury patients Clinical urine specimens were col-lected from brain injury patients upon admission to acute brain injury This is among the unique studies that have assessed the least invasive approach (urine specimens) injury indices and correlated

ele-to the protein interacele-tome and the altered pathways (outgrowth and guidance, extracellular matrix, postsynaptic density proteins, etc.) Interestingly, these neuroproteomics data were correlated to

one study by Ren et al., intracerebral hemorrhage (ICH) was assessed via neuroproteomic approach (LC-MS/MS) studying altered expression of proteins that are induced in brain tissue 3 h after injury in a rat model of ICH This is among the recent studies that utilized systems biology approaches to discern the function of different proteins indicating that altered proteins fell in the catego-ries of autophagy regulation, ischemia, necrosis, apoptosis, calpain

7 Recent Application in Neuroproteomics 3D MALDI Imaging

Another interesting area of neuroproteomics approaches is the duction of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) known as MALDI- IMS The area

intro-of MALDI-IMS is a novel proteomic tool that provides an overview depiction of the overall distribution and localization for different

simultaneous mapping of hundreds of peptides and proteins present

can be seen as a complementary approach to immunohistochemistry

MALDI-IMS images enable the spatial spread of a particular peak’s height retaining the histopathological context where the analyte sig-nals are correlated with underlying tissue architecture without any

dis-tinguishing feature of MALDI-IMS from other MS techniques is that the preparation of the sample and the acquisition of the MS data are performed to keep the sample spatial integrity along the limits of

the methodology and application on MALDI-IMS has been

Reconstructed images represent a visual representation of the biological sample achieved by plotting the intensities of a given ion

on a coordinate system that represents the relative position of the

the use of the brain tissue was often the organ of choice for method development in MALDI-IMS due to the inherent brain bilateral

symmetry providing an internal quality control for ion image MALDI-IMS is being used to study the pathological processes

In his work, Shanta et al assessed levels of membrane pholipids affected by oxidative stress postischemic injury Thus, altered lipid components were evaluated during ischemia utilizing MALDI-IMS technique to identify phospholipids profile between damaged and normal brain regions Several phospholipids such as lysophosphatidylcholine, phosphatidylcholine, phosphatidyletha-nolamine, and sphingomyelin were identified by MALDI-IMS which followed by MS/MS analysis Several of these phospholipids were considered as therapeutic targets for ischemic intervention

the glycosphingolipid family, enriched within the central nervous system and are involved in brain disease development, were assessed spatially following mouse middle cerebral artery occlusion (MCAO)-reperfusion injury which was performed using MALDI- IMS technique Of interest, there was a marked variability in the ratio of expression between ipsilateral and contralateral cortices in the ganglioside species expression post-MCAO-reperfusion injury Most interestingly, MCAO resulted in the transient induction of both GM2 and GM3 signals within the ipsilateral hemisphere

Along the same line, Koizumi et al performed IMS analysis on rat brain tissue sections with focal cerebral ischemia in rat model

cerebral artery occlusion, and brain sections were prepared Several species were identified including phosphatidylcholine and lyso-phosphatidylcholine, which were altered post-cerebral ischemia

et al used MALDI-IMS to assess alterations in ganglioside species (GD1a, GM1, GM2, and GM3) in the presence of beta-amyloid

Alzheimer’s disease), and combined exposure Data showed that GM2 and GM3 are involved as a common culprit in the interactive

matrix of silver nanoparticles was implanted on brain sections for MALDI-IMS across unfixed cryostat sections of rat brain post- controlled cortical impact injury Brain lipid composition was assessed in the brain section Of interest, in the ipsilateral area, ceramides and decreased sphingomyelins, accompanied by changes

in glycerophospholipids and cholesterol derivatives, were observed which occurred in a spatial distribution of 1, 3, and 7 days This neuroproteomics approach exhibited features for revealing unde-tectable cellular injury response that can be used as a new index for

Crecelius et al., 3D reconstruction of myelin basic protein (MBP)

was performed in the mouse brain corpus callosum where optical images from serial coronal sections reconstructed a surface of the corpus callosum MALDI-IMS data were superimposed to create the final 3D visualization Of interest, the work by Crecelius et al

is considered among the best examples where proteomic data can

be correlated with anatomical structures providing depth analysis

Finally, in a very elegant study by Devaux et al., 3D matrix- assisted laser desorption/ionization (MALDI) imaging was uti-lized to depict the spatiotemporal proteome molecular kinetics post-acute spinal cord injury in rat model This technique assessed the rostral and caudal segments, which were compared to the lesioned area 3 days, post-SCI representing a novel approach to define new dimension of neuroproteomics platforms addressing lipid reorganization in the white matter region of injured spinal

8 Concluding Remarks in Neuroproteomics Research

In this work, we summarized the recent output of the teomics discipline focusing on its applications in the area of neu-rotrauma As we have seen that genomics cannot completely answer all the questions that arise in studying the nervous system Indeed, in a variety of fields, scientists criticize the use of genomics

neuropro-as a tool, because DNA sequencing provides only a snapshot of the different ways a cell may use its genes Any cell constantly reacts to its changing environment differently, creating a dynamic system

between changes at the transcription level and changes at the

well; thus, highlighting the capabilities of different teomics in different applications

neuropro-To conclude, neuroproteomics approaches have been fully applied in several brain-related disciplines to understand brain functions in conjunction with neurosystems biology approaches

whole or part of the neuroproteome to interrogation It describes how proteins are dynamically regulated and altered in terms of expression, modification, and translation To this end, the out-come of proteomics is positive; however, proteomics studies pro-posed in any proposal or grant are being often described as being

“fishing expeditions,” i.e., hypothesis-free with no defined aim or endpoint In other words, the write-up dictates the theory that we will search for protein changes and then we will provide the scien-tific questions However, this concept can be reversed proving that proteomics studies can be hypothesis driven requiring defined, critical, and correct questions to be asked This requires focusing

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Chapter 2

Progress and Potential of Imaging Mass Spectrometry

Applied to Biomarker Discovery

Jusal Quanico, Julien Franck, Maxence Wisztorski, Michel Salzet,

and Isabelle Fournier

Abstract

Mapping provides a direct means to assess the impact of protein biomarkers and puts into context their relevance in the type of cancer being examined To this end, mass spectrometry imaging (MSI) was devel- oped to provide the needed spatial information which is missing in traditional liquid-based mass spectro- metric proteomics approaches Aptly described as a “molecular histology” technique, MSI gives an additional dimension in characterizing tumor biopsies, allowing for mapping of hundreds of molecules in

a single analysis A decade of developments focused on improving and standardizing MSI so that the nique can be translated into the clinical setting This review describes the progress made in addressing the technological development that allows to bridge local protein detection by MSI to its identification and to illustrate its potential in studying various aspects of cancer biomarker discovery.

tech-Key words Mass spectrometry imaging, Molecular histology, Biomarker, Protein identification,

Microextraction, Matrix-assisted laser desorption/ionization

1 Introduction

MSI is a technique used to map the distribution of various classes

Discrimination of the specific distribution of these molecules allows for MSI to be used in identifying diseased regions within tissue sections, making MSI a suitable technique for examining tumor biopsies Using statistical methods, signals of molecules defining these diseased regions can be extracted and used to generate mod-els that can serve as diagnostic indicators of the disease and as pre-dictors of disease outcome and patient survival Among the lead candidates obtained using the MSI approach, particular emphasis has been put in the identification of protein biomarkers Unlike lipids and metabolites, proteins are direct translation products of genetic information coded in DNA Posttranslational modifications

further highlight the relevance of proteins in biomarker discovery studies, because such information reflect disease-associated changes that are not observed in other biomolecules particularly RNA.

way from the development of the technique (including

computational methods in order to extract pathology-relevant

tech-nique on large-scale biomarker discovery studies involving

compre-hensive detail And yet, many fundamental aspects of the nique, such as detection sensitivity, spatial and spectral resolution,

tech-in situ identification, speed of analysis, and method reproducibility, continue to impose challenges that need to be surmounted prior to its widespread implementation in the clinical setting To discuss these points, the present review is divided into three main sections The first section describes the progress made toward addressing some of these issues, with particular emphasis on efforts to improve protein detection and identification In the second section, recent MSI-related applications in cancer are also presented, demonstrat-ing the potential of the method in this field even at the current stage of its development Finally, we describe MSI-guided micro-extraction strategies to bridge the gap between LC-MS-based bio-marker discovery pipelines and MSI in an effort to further address the current limitations of MSI

2 MSI Challenges for Clinical Application

The detection of proteins by MSI remains a challenge, particularly for less abundant proteins This is primarily a consequence of the limited number of copies of the protein expressed per unit cell rela-tive to the abundance of other compounds present (metabolites, lipids, and endogenous salts) and sensitivity of the current instru-mentation, compounded by the ion suppression effect In MSI, detection depends on the amount of cells sampled in one raster spot and is dictated by the diameter of the laser used Thus, as the lateral resolution is increased by performing detection at microm-eter and sub-micrometer laser diameters, concomitant decreases in

dependent nature of spectral sampling in MALDI, it is difficult to perform protein enrichment and pre-concentration prior to MS acquisition without compromising to some extent their localiza-tion on the tissue Also, depending on the nature of the sample, the efficiency of the sampling probe can vary, further limiting pro-tein detection sensitivity

2.1 Protein Detection

As protein therapeutic targets often fall in the category of less abundant proteins, it is crucial that improvements in protein detec-tion sensitivity be addressed before MSI can fully be utilized as a diagnostic tool for pathologies In recent years, MSI groups have attempted to address this limitation in different ways These can be classified as improvements in terms of sample preparation steps (choice and deposition of matrix, sample treatment, and analyte conversion) and data acquisition (focusing on ion detection).

In contrast to lipids, proteins need to be incorporated into the matrix crystals for them to be efficiently desorbed and ionized in MALDI This entails that during matrix deposition, a sufficient amount of time must be provided to allow the crystals to form and grow However, the size of the crystals must be controlled in order

to keep a homogeneous matrix deposition throughout the tissue section, as the crystal size influences spatial resolution and mini-mizes protein delocalization In addition, the choice of solvent is also crucial, as the solvent facilitates analyte extraction and, depend-ing on its volatility, the crystallization of the matrix Thus, interplay between these factors must be optimized in order to improve pro-tein detection while keeping its localization information preserved.One strategy to improve protein extraction and consequently detection is by microspotting the matrix This involves deposition

of picoliter quantities of the matrix using a piezoelectric chemical inkjet printer, an acoustic robotic spotter, or a modified LC

down to picoliter quantities, these methods restrict analyte calization to the size of the spot deposited without compromising the volume of solvent delivered per spot As the volume of solvent delivered is relatively larger compared to other matrix deposition methods, extraction efficiency using this approach remains high

delo-A recent demonstration of the use of microspotting in MSI has

spotter was used to assess the limits of detection of intact proteins during an MSI experiment In this work, protein standards were deposited onto the tissue surface using an acoustic robotic spotter, and the tissue was dried prior to matrix deposition Results led to the detection of micromolar to millimolar quantities of standard proteins with the values higher for experiments involving on-tissue trypsin digestion With these low limits of detection, the authors have illustrated through their systematic methodology that a significant improvement in protein detection must be developed in order for MSI to be able to detect proteins of significant therapeu-tic importance

Although microspotting allows for a more sensitive detection

of proteins by MSI, the approach is limited in lateral resolution achievable to several hundred micrometers (typically between 100

2.1.1 Matrix

that have to be maintained to prevent intermingling of the vidual spots Notably at high-resolution MSI, however, such a matrix deposition method becomes insufficient in providing homo-geneous matrix deposition Another approach involves uniform deposition of the matrix with minimal use of solvent, which can be achieved by various methods (such as spray coating and vibrational vaporization) Uniform spray coating can be achieved using com-mercially available devices such as the TM sprayer and the SunCollect instrument However, for such devices, control of tis-sue wetness during spraying is difficult and requires sample-tailored optimization prior to use Vibrational vaporization using the ImagePrep instrument allows the controlled deposition of matrix

The instrument uses a light-scattering detector to measure the thickness of the matrix deposited on the sample surface and more crucially the wetness of the tissue section Efficient control of the nebulization and incubation parameters enables efficient extraction

of proteins and incorporation into the matrix crystals

In spite of the notable advantages in using vibrational ization devices, recent developments in high-resolution MSI pre-cluded the use of solvent-free matrix deposition methods to improve spatial resolution in these applications Solvent-free matrix deposition by sublimation followed by recrystallization under con-

espe-cially with the use of organic solvent mixtures with water allows for extended periods of vapor exposure of the matrix and thus better integration of the analytes without forming large matrix crystals Recently, matrix pre-coating followed by treatment with di- isopropylethylamine and water vapor has been demonstrated to

their previous work on this subject

In contrast to the aforementioned approaches, a recent tigation instead attempted to enhance the migration of analytes

coating assisted by an electric field (MCAEF), the technique applied an electric field during matrix deposition in an ImagePrep instrument to stimulate the migration of positively and negatively charged analytes, doubling the detection and imaging of both lip-ids and proteins (at 3.5–37 kDa)

Tissue preparation for MSI analysis is one of the most crucial stages for the improved detection of proteins Washing of tissues, for example, together with the proper choice of matrix significantly alters the type of analyte that will be observed in MALDI MS In the case of proteins, depletion of lipids and salts by washing using several grades of ethanol followed by chloroform has been effective

also be tailored to suit particular samples For example, several

2.1.2 Tissue Treatment