Essential Techniques for Medical and Life Scientists: A guide to contemporary methods and current applications with the protocols: Part 1 E-Book

• Herausgeber: Bentham Science Publishers
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 213

Veröffentlichungsjahr: 2018

Beschreibung

This handbook covers some primary instruments-based techniques used in modern biological science and medical research programs. Key features of the book include introductory notes for each topic, systematic presentation of relevant methods, troubleshootin

Leseprobe

Table of Contents

Table of Contents

Title

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FOREWORD

PREFACE

List of Contributors

Application of Mass Spectrometry in Proteomics

Abstract

INTRODUCTION

1. Inlet Systems

2. Ionization Technique

2.1. Electrospray Ionization (ESI)

2.2. Matrix Assisted Laser Desorption Ionization (MALDI)

2.2.1. MALDI Sample Preparation

3. Mass Analyzers

3.1. Quadrupole Mass Analyzer

Tandem Mass Spectrometry MS/MS

3.2. Ion Trap Mass Analyzer (IT)

3.3. Orbitrap Mass Analyzer

3.4. Time of Flight (TOF) Mass Analyzer

4. Detectors

5. Applications

5.1. Proteomics

5.1.1. Molecular Weight Determination

5.1.2. Protein Sequencing

5.1.2.1. Sample Preparation for Protein Sequencing

5.1.3. Post-Translational Modifications

5.1.4. Protein-Protein, Protein-DNA and Protein-Small Molecule Interactions

5.1.4.1. Hydrogen Deuterium Exchange (HDX) Coupled with MS (HDX-MS)

EX1 and EX2 Regimes

5.1.4.2. Chemical Cross Linking Coupled by MS (XL-MS)

5.1.5. Protein Folding and Unfolding Rates

5.2. Microbiology

5.3. Medicine

CONSENT FOR PUBLICATION

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Structural Elucidation of Macromolecules

Abstract

INTRODUCTION

2.1. Solving the 3D Structure of Proteins Using X-ray Crystallography

2.1.1. Protein Crystallization

2.1.2. Diffraction of X-rays by Protein Crystals

2.1.3. 3D Structure Solution and Refinement

2.1.4. Validation and Analysis of Crystal Structures

2.2. NMR Guide into Molecular Recognition

2.2.1. NMR and the Study of Intermolecular Interactions

2.2.2. Protein-detected NMR Based Experiments

2.2.2.1. Chemical Shift Perturbation

2.2.3. Ligand-detected NMR Based Experiments

2.2.3.1. NOE-based Methods

Transferred NOE

2.2.3.2. Diffusion Experiments

2.3. Studying Protein Shape and Protein-ligand Interactions by SAXS Methods

2.3.1. Data Collection

2.3.2. Preliminary Sample Characterization: Guinier; Porod, Kratky and Pair Distribution Functions

2.3.3. Deriving a Structural Model

2.4. Structural Elucidation Using Cryo-Electron Microscopy

2.5. Notes

2.5.1. Growing and Preserving a Protein Crystal

2.5.2. Bragg’s Law

2.5.3. Seleno-methionine Derivatives for Structure Solution

2.5.4. Patterson Maps and Molecular Replacement

2.5.5. Electron Density Maps

2.5.6. The Temperature Factor (B Factor)

2.5.7. The Ramachandran Plot

2.5.8. Technical Aspects of NMR Hardware and Software

2.5.9. NMR Sample

2.5.10. General Aspects on Acquisition and Processing of NMR Spectra

2.5.11. Basics on 15N-1H-HSQC Titrations

2.5.12. Basics on Transferred NOE

2.5.13. Basics on STD-NMR Experiments

2.5.14. Basics on Diffusion Experiments

2.5.15. SAXS Buffer Subtraction

2.5.16. Sample Quality for Successful BioSAXS Experiment

2.5.17. Radiation Damage

2.5.18. Small Angle Neutron Scattering (SANS)

CONSENT FOR PUBLICATION

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

References

Isothermal Titration Calorimetry

Abstract

INTRODUCTION

3.1. ITC in Protein Science and Enzyme Technology

3.1.1. Protein-protein Interactions

3.1.2. Protein- DNA/RNA Interactions

3.1.3. Enzyme Kinetics

3.2. ITC in Pharmaceutical Research

3.2.1. ITC in Drug Delivery

3.2.2. ITC in Drug Screening

3.2.3. ITC in Drug Design

3.2.4. ITC in Drug Development

3.3. ITC Applications in Food Science

3.3.1. Interactions Involving Proteins or Peptides

3.3.1.1. Interactions Between Flavan-3-ols and L-proline

3.3.1.2. Interactions Between Green Tea Flavanoids and Milk Proteins

3.3.1.3. Interactions Between Soy Protein and Acid Mineral Solution

3.3.1.4. Interactions Between Epicatechin and Serum Albumin

3.3.2. Interactions Involving Carbohydrates

3.3.2.1. Carbohydrate Binding Properties of Banana Lectin

3.3.2.2. Interactions Between Chitosan and Bile Salt

3.3.3. Interactions Involving Lipids

3.4. Protocol

3.4.1. Sample Preparation

3.4.2. Measurement

3.4.3. Data Collection and Analysis

3.5. Troubleshooting

CONSENT FOR PUBLICATION

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Abstract

INTRODUCTION

4.1. Methodology

4.1.1. Isolation of RNA

4.1.2. Reverse Transcription or Complementary DNA Synthesis (cDNA Synthesis)

4.1.3. PCR Amplification

4.1.3.1. Designing Primer

4.1.3.2. PCR Amplification and Detection

4.2. Applications

4.3. Precautions to be Taken

4.3.1. Real-time Quantitative PCR (RT-qPCR)

4.4. Future Perspectives

CONSENT FOR PUBLICATION

CONFLICT OF INTEREST

ACKNOWLEDGEMENTS

REFERENCES

Essential Techniques for Medical and Life Scientists:

 A Guide to Contemporary Methods and Current Applications with the Protocols

(Volume 1)

Edited by

Yusuf Tutar

University of Health Sciences,

Istanbul,

Turkey

BENTHAM SCIENCE PUBLISHERS LTD.

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FOREWORD

Evaluating hypotheses and making judgements on the basis of well supported evidence solve problems in science. Medical and biological data mainly consist of measurement of assay conditions to support the proposed hypothesis. A prediction must test variables and scientist must acquire data from experiments to elicit an explanation or description to the scientific problem. Experimental results of the assay provide direct or indirect evidence to the theory. Therefore, several techniques have been developed to help researchers facilitate experimental design and to elucidate molecular mechanism of biological systems. Some techniques alone may provide unique evidence while still other complement other techniques. For this reason, investigators must be informed about the techniques and know them in detail. It is hard to follow up each technique specially for early career scientists and its applications for experienced researchers. The first volume of this book provides a training platform and application modules of each technique and may help individual investigators to guide research practices.

Prof. Lütfi Tutar Ahi Evran University, Turkey

PREFACE

This book focuses on instrumental techniques and their applications in medicine and biological sciences. Chapter 1 discusses mass spectroscopy (MS) and this technique provides qualitative and quantitative measurements of biological samples. MS has several applications but have not been mainly employed in proteomics studies yet. However, recent developments in clinical applications of MS courage researchers to perform difficult assays precisely and rapidly.

Structural elucidation of macromolecules form the basis of molecular biophysics and Chapter 2 discusses X-ray crystallography, Nuclear Magnetic resonance (NMR), Small Angle X-ray scattering (SAXS), and Cryo-electron Microscopy. Without detailed structure of a macromolecule, it is hard to understand the macromolecule interaction in biochemistry. So far, these methods have been employed in biological systems to reveal macromolecule structures. Each method has a unique site as well as overlapping functions however, all of them are useful to structural biologists, pharmaceutical, and medical scientists.

Chapter 3 explains a unique technique; isothermal titration calorimetry (ITC). It is unique since the technique not only measures binding affinity but also it provides thermodynamic data. The thermodynamic data provides nature of interaction. ITC measures any interactions between different macromolecules, DNA, RNA, lipid, carbohydrate, protein (enzyme, antibody), and ligand of any type. The component of the assay may be more than two molecules and there is no molecular weight restriction for the assay. Since it measures heat differences as signal, opaque solutions and suspensions do not restrict measuring binding affinity. ITC also measures enzyme kinetics and the technique may also be coupled to spectroscopic techniques like fluorescence. ITC is an all in one instrument for scientist. The chapter discusses applications of ITC in different disciplines.

Chapter 4 describes a common instrument of life science laboratory; reverse transcription polymerase chain reaction. This powerful method has found applications in medical, diagnosis, and forensics. Methodology and applications of the technique are explained thoroughly in this chapter.

This book is designed not only for early career young scientists (graduate students or postdoctoral associates) but for scientists who are experts in a particular technique but want to use different applications for their experimental set up. Next volume of the book will provide chapters for different analytical techniques.

Dr. Tutar would like to acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”.

Prof. Yusuf Tutar University of Health Sciences, Istanbul Turkey

List of Contributors

Ana Luísa CarvalhoUCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalBanu BayramHealth Sciences Faculty, Nutrition and Dietetics Department UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalElvan Yılmaz AkyüzHealth Sciences Faculty, Nutrition and Dietetics Department Health Sciences Faculty, Nutrition and Dietetics Department Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, IndiaEsen TutarKahramanmaraş Sütçü Imam University, Science and Letters Faculty, Avsar Campus, 46060, Kahramanmaras, TurkeyEurico J. CabritaHealth Sciences Faculty, Nutrition and Dietetics Department UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalFilipa MarceloUCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalHalime Hanım PençeFaculty of Medicine, Division of Biochemistry Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, IndiaMaria João RomãoUCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalÖzlem AytekinHealth Sciences Faculty, Nutrition and Dietetics Department Health Sciences Faculty, Nutrition and Dietetics Department Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, IndiaSathishkumar RamalingamFaculty of Medicine, Division of Biochemistry Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, IndiaSerap PektasDepartment of Chemistry, Recep Tayyip Erdogan University, Rize, TurkeyTeresa Santos-SilvaUCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, PortugalVenkidasamy BaskarHealth Sciences Faculty, Nutrition and Dietetics Department Health Sciences Faculty, Nutrition and Dietetics Department Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, IndiaYusuf TutarFaculty of Pharmacy, Division of Biochemistry University of Health Sciences 34668, Istanbul, Turkey

Application of Mass Spectrometry in Proteomics

Serap Pektas*

Department of Chemistry, Recep Tayyip Erdogan University, Rize, Turkey

Abstract

Mass spectrometry (MS) is a powerful tool to study biological samples both qualitatively (structure) and quantitatively (molecular mass). In recent years with the improvement of soft ionization techniques and the development of new methods, its application in proteomics, microbiology and clinical laboratories has increased. Especially in proteomics laboratories, it is commonly used for determining protein amino acid sequence, identification of post translational modifications, determining protein-peptide/protein/DNA interactions, determining protein folding and unfolding rates etc. In microbiological laboratories, MS is mainly used to identify microorganisms such as bacteria and fungi. Even though its use in clinical laboratories still needs improvement of methods, it can be used for diagnosis of disease, identification of metabolic disorders, discovering new biomarkers and identifying drug toxicity. This chapter provides a general review of MS applications in proteomics.

Keywords: Chemical Cross Linking coupled by MS (XL-MS), Electrospray Ionization, Hydrogen Deuterium Exchange coupled by MS (HDX-MS), Ion Trap Mass Analyzer, Matrix Assisted Laser Desorption Ionization, Proteomics, Protein Sequencing, Quadrupole Mass Analyzer, Time of Flight Mass Analyzer.

*Corresponding author Serap Pektas: Department of Chemistry, Recep Tayyip Erdogan University, Rize, Turkey; Tel: [phone] (1819); E-mail: [email protected]

INTRODUCTION

The sample of interest is introduced to the mass spectrometer using an inlet system then reaches into the ionization source, where they are ionized. The formed ions are then arrived to a mass analyzer. After the ions reach to the mass analyzer, they separated based on their mass/charge (m/z) ratios later transferred to a detector. Finally signals are recorded by a computer system. The signals displayed as a mass spectrum showing the relative abundance of signals according to their m/z ratios. A typical mass spectrometer consists of the following four main parts (Fig. 1):

Fig. (1)) Mass spectrometer parts. Inlet system (LC, GC, Direct Probe)Ion source (ESI, MALDI, FAB, CI, EI)Mass analyzer (Quadrupole, Time of Flight (TOF), Ion Trap, Magnetic Sector)Detector (Electron Multiplier, Micro Channel Plates MCPs)

1. Inlet Systems

The function of an inlet system is to introduce a small amount of sample into the ion source. Sample can be injected to the mass spectrometer with different ways depend on the nature of the sample. One of them is batch inlet system, which involves the volatilization of sample externally, then gradually leakage of the volatilized sample into the ionization source. It is not suitable for the liquid samples that have boiling temperature of 500 oC [1]. Another inlet system is called direct probe inlet. Direct probe inlet is suitable for solid and nonvolatile liquids [2]. If the analyte is a mixture then sample can be introduced using one of the chromatographic techniques like liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) [2, 3]. The given name of a MS instrument may refer to inlet system that a mass spectrometer has, such as LC-MS, GC-MS and CE-MS.

2. Ionization Technique

MS measures the masses of ions and therefore sample has to be ionized to be able to measure its mass. There are multiple ion sources and each of them has their own advantages and disadvantages depending on the analyte of interest. Ionization techniques used in MS are listed below:

Fast Atom Bombardment Ionization (FAB)Electrospray Ionization (ESI)Matrix Assisted Laser Desorption Ionization (MALDI)Electron Ionization (Electron Impact Ionization) (EI)Chemical Ionization (CI)Native Ion Chemical Ionization

Depending on the chemical and physical properties of the sample of interest, different ionizations techniques can be used. One of the main factors for choosing the most suitable ionization source is the thermolability of the analyte. For non-thermolibale and volatile samples, electron ionization and/or chemical ionization techniques can be used. However, thermally liable and nonvolatile samples such as peptides, proteins, and other biological samples, softer ionization techniques are more suitable. The most common soft ionization sources used for the biological samples are ESI and MALDI [4, 5]. Therefore, these ionization techniques will be discussed in this chapter. The given name of a mass spectrometry technique is usually refers to the ionization method being used such as ESI-MS and MALDI-MS.

2.1. Electrospray Ionization (ESI)

Electrospray ionization (ESI) technique is one of the softest ionization techniques in MS. In the past few decades, it became an important technique in structural biology laboratories and clinical laboratories for qualitative and quantitative measurement of metabolites in a complex mixture of sample. Basic principle of ESI is outlined in Fig. (2), ESI technique can be divided into three main steps. The first step is the nebulization of the sample solution into electrically charged droplets. The second step is the release of ions from droplets and the final step is the transportation of ions to the mass analyzer [6-8].

Fig. (2)) Mechanism of Electrospray Ionization.

Sample solution passes through a capillary tube at a high voltage to generate ions. The applied potential between capillary tube and counter electrode is usually between 2.5-6 kV. After the initial formation of electrically charged droplets, they shrink in size by the evaporation of solvent, with the help of an ESI source temperature and/or nitrogen drying gas (nebulizer gas). The evaporation of solvent leads to a high charge density of droplets and coulomb repulsion force. When the electrostatic repulsion becomes stronger than the surface tension smaller electrically charged droplets are formed. Eventually ions at the surface of a droplet get ejected into the gaseous phase and then ions got accelerated into the mass analyzer [6]. However, for larger molecules like proteins, another model called charged residue mechanism is widely accepted. In this mechanism, due to the solvent evaporation and coulomb repulsion force, a very small charged droplet containing only a single analyte molecule is formed. After the desolvation of the charged droplet, its charge retains on the analyte molecule.

In ESI, technique charging is due to extra protons on analyte (or the loss of protons in negative mode) and compounds needs to have an acidic or basic charge to be ionized. One of the main advantages of ESI technique is that multiple charges can be generated which allow the determination of big molecules such as peptides and proteins.

Advantages:

It is a very gentle ionization technique therefore it is suitable for biological molecules (proteins, peptides, etc.)Can analyze very large moleculesVery sensitive and very efficient ionization techniqueSuitable to couple with LC systems.

Disadvantages:

Low ionic strength solutions are necessary otherwise, the detector can be blocked and instrument may require maintenance (for protein and peptides only volatile buffers such as ammonium acetate can be used). But using low ionic strength solution may cause stability problems with some protein samples.Detergents are also not recommended because they suppress the ionization of analytes.It runs as continuous flow, which causes relatively more sample consumption compared to MALDI.

2.2. Matrix Assisted Laser Desorption Ionization (MALDI)

MALDI is a soft ionization technique used in MS. Thus, it can be used in the analysis of biomolecules such as peptides, proteins, DNA and sugars. In this technique, sample is co-crystalized with a UV absorbing substance called matrix (usually organic acids with conjugated pi system) [5, 9]. Ionization with MALDI requires three-step process. Initially, the sample is co crystalized with a suitable matrix and applied to a MALDI target (sample plate). Second, a laser pulse is used to irritate and trigger desorption of the analyte. Finally, the analyte molecules get ionized (Fig. 3).

Fig. (3)) Schematic diagram of a MALDI system.

Even though ionization mechanism by MADLI has not been well understood, matrix serves three main purposes. First, it absorbs laser energy and controllably transfers the energy. Second, it provides surface charging and finally keeps the analyte from aggregating. Table 1 shows the molecule of interests and appropriate matrix to use. As a laser source, nitrogen laser (337 nm) and frequency tripled and quadrupled Nd:YAG lasers (355nm and 266 nm, respectively) are commonly used [9]. Even though it is not very common, infrared lasers are also used in MALDI. One characteristic of MALDI is, that it commonly generates singly protonated molecules (M + H)+.

Table 1Molecule of interest and appropriate matrixes [9].Molecule of InterestMatrixAbbreviationPeptides/Proteins Mass < 10 000 Mass > 10 000α-Cyano-4-hydroxycinnamic acid Sinapic acid 2,5-dihydroxybenzoic acidCHCA SA HABANucleotides Mass < 3.5 kDa Mass > 3.5 kDa2,4,6-Trihydroxyacetophenone 3-Hydroxypicolinic acid Anthranilic acid Nicotinic acid SalicylamideTHAP HPALipidsDithranolDITCarbohydrates2,5-Dihydroxybenzoic acid α-Cyano-4-hydroxycinnamic acid 2,4,6-Trihydroxyacetophenone 3-AminoquinolineDHB CHCA THAP

2.2.1. MALDI Sample Preparation

Sample preparation can be divided into the following steps:

Initially a solution is prepared using acetonitrile (ACN) (or methanol), water and trifluoroacetic acid (TFA) (or formic Acid, FA). The ratio of the solvent varies but 50:50:0.2 (ACN:Water:TFA) ratio is very common.After ACN:Water:TFA solution prepared, a saturating amount of MALDI matrix is added into the solution (it has to be saturated).Sample of interest is then added into the MALDI matrix solution and mixed well (with the help of a vortex).Then, 1-2 μL of matrix-protein mix is applied on MALDI target (sample plate). Finally, samples can be analyzed by MALDI-MS after samples are air-dried or blow-dried.

Advantages:

A fast ionization techniqueLess sample loss due to the non-continuous flowMore tolerant to salt concentration than ESI

Disadvantages:

Non-continuous nature of the technique makes it difficult to couple with LC systemsNot very convenient to perform protein MS/MS (for now)Not suitable for HDX-MS experiment due to the difficulty with temperature control (see section 5.1.4.1 of this chapter).

3. Mass Analyzers

A mass analyzer separates the ionized masses based on mass to charge (m/z) ratios, then a detector measures the signal. There are six types of mass analyzers that can be used in MS, each for different purposes. Among these mass analyzers, the following first four are commonly used in biological laboratories and they will be discussed in more details.

Quadrupole Mass AnalyzerIon Trap Mass AnalyzerOrbitrap Mass AnalyzerTime of Flight Mass AnalyzerMagnetic Sector Mass AnalyzerElectrostatic Sector Mass AnalyzerIon Cyclotron Resonance

3.1. Quadrupole Mass Analyzer

The quadrupole mass analyzer is one of the most commonly used mass analyzer in mass spectrometers. They are robust, economical, small size mass analyzers and suitable with different inlet systems.

Fig. (4)) Schematic diagram of a quadrupole mass analyzer.

Quadrupole system consists of 4 parallel metal rods placed in equal distance and each opposite rods connected with DC (direct current) and RF (radio frequency) voltages (Fig. 4). The both voltages create an electrical field, which causes ions to travel with oscillatory motion in the Z direction. The oscillatory motion’s amplitude is related with ions m/z ratio and allows selective transmission of ions [10-12]. By changing DC and RF voltages ions passing to detector can be controlled. If the ions are off the set m/z ratio limits, they hit the metal rods and cannot reach to the detector [10-12]. A mass spectrometer may have more than one quadrupole mass analyzer, for example, a tandem mass spectrometer may contain three quadrupole mass analyzers.

Tandem Mass Spectrometry MS/MS

A tandem mass spectrometry is applying two stages of mass analysis. It is also known as MS/MS or MS2 (Fig. 5). A tandem mass spectrometer has more than one mass analyzer, which allows the analysis of analyte at a structural level [2, 12-14].

Fig. (5)) Tandem MS system.