PROTEOMICS IN PERSONALIZED MEDICINE: TAILORING

TREATMENT TO INDIVIDUALS

BY

ADETONA, TIMILEYIN SAMUEL

(2020/29901)

A SEMINAR REPORT SUBMITTED TO THE DEPARTMENT OF

BIOCHEMISTRY,

FACULTY OF BASIC AND APPLIED SCIENCES OSUN STATE

UNIVERSITY, OSOGBO

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE

AWARD OF BACHELOR OF SCIENCE (B.SC. HONS) DEGREE IN

BIOCHEMISTRY

FEBUARY, 2023
 CERTIFICATION

I hereby certify that ADETONA TIMILEYIN SAMUEL (2020/29901) wrote this seminar

under my supervision and it has been read and approved as meeting part of the requirement for

the award of Bachelor of Science (B.Sc. Hons) Degree in Biochemistry, Osun State University,

Osogbo.

……………………………….. …………………………

Mr. E.A. Iyoha Date

Supervisor

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 ACKNOWLEGEMENTS

I am grateful to God Almighty for being the source of my inspiration, wisdom, and

understanding. I will forever be thankful for the greatest opportunities he has given to me. I

will also wish to acknowledge the effort of my parent, Mr. and Mrs. Adetona, my siblings for

their valuable insights, guidance, towards the achievement of my academic pursuit, their

encouragement greatly enriched the content of this seminar report. I will never hold back to

acknowledge the efforts of the lecturers in the department, they have been a carrier and

supporter towards the growth Myself, May God’s blessings be sufficient for you and your

families.

To my supervisor, Mr. E.A. Iyoha, I really appreciate you for your effort towards the success

of my seminar, and to the generality of Biochemistry Department, I say thank you all.

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 TABLE OF CONTENTS

PAGE

Certification…………………………………………………………………………………..i

Acknowledgements..…………………………………………………………………………ii

Table of Content……………………………………………………………………………..iii

List of Tables…………………………………………………………………………………v

List of Figures………………………………………………………………………………..vi

CHAPTER 1: INTRODUCTION....…………………………………………………….....1

1.1 INTRODUCTION TO PROTEOMICS………………………………………………...1

1.2 INTRODUCTION TO PERSONALIZED MEDICINE………………………………..4

1.3 RELATIOSHIP BETWEEN PROTEOMICS AND PERSONALIZED MEDICINE….5

CHAPTER 2: PROTEOMICS TECHNIQUES…………………………………………...7

2.1 PROTEOMICS WORKFLOW……………………………………………………….....7

2.2 SEPARATION AND ISOLATION OF PROTEIN……………………………….……10

2.2.1 GEL-BASED APPROACH……………………………………………………...10

2.2.2 CHROMATOGRAPHY-BASED APPROACH…………………………………11

2.3 PROTEIN IDENTIFICATION AND CHARACTERIZATION…………………….....12

2.3.1 MASS SPECTROMETER………………………………………………………13

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 2.3.2 EDMAN SEQUENCE…………………………………………………………..16

2.3.3 PROTEIN MICROARRAY……………………………………………………..16

2.4 PROTEIN IDENTIFICATION AND VALIDATION…………………………………18

2.5 BIOINFORMATICS IN PROTEOMICS………………………………………………18

CHAPTER 3: APPLICATIONS OF PROTEOMICS….………………………………...20

3.1 BIOMARKER DISCOVERY…………………………………………………………..20

3.2 DRUG DISCOVERY AND DEVELOPMENT…………………………...…………...24

3.3 ONCOLOGY…………………………………………………………………………...25

3.4 PERSONALIZED PROTEOMICS……………………………………………………..31

3.4.1 PROTEOMICS IN DIAGNOSIS………………………………………………..31

3.4.2 PROTEOMICS TO IMPROVE PATIENT/TREATMENT SELECTION……..33

CHAPTER 4: CONCLUSION………………………………………………………...…..35

4.1 THE FUTURE OF PROTEOMICS……………………………………………………36

REFERENCE……..………………………………………………………………………..39

APPENDIX…………………………………………………………………………………42

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 LIST OF TABLES
Table 3.1: Table showing different quantification methods with their advantages and

disadvantages (Kwon, Y. et al. 2021)……………………………….………………………...30

v
 LIST OF FIGURES

Figure 2.1 Description of Top-down and Bottom-up workflow of proteomics (Beeton-

Kempen, N. , 2020)……………..………………………………………….………..………....9

Figure 2.2 Matrix-Assisted Laser Desorption/Ionization and Electrospray Ionization (Beeton-

Kempen, N. , 2020) ……………………………………………….………………………….15

Figure 2.3 Protein microarray (Beeton-Kempen, N. , 2020)……………………..…………..17

Figure 3.1 Steps involved in proteomics analysis (Kwon, Y. et al. 2021)……….……………27

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 CHAPTER ONE

INTRODUCTION

1.1 INTRODUCTION TO PROTEOMICS

Proteomics is a new type of ‘omics’ that has rapidly developed, especially in the therapeutics

field. The word proteome was created by Marc Wilkins in 1995. Proteomics is the study of the

interactions, function, composition, and structures of proteins and their cellular activities.

Proteomics provides a better understanding of the structure and function of the organism than

genomics. However, it is much more complicated than genomics because the protein

expression is altered according to time and environmental conditions. It is estimated that there

are almost one million human proteins, many of which contain some modifications such as

post-translational modifications (PTMs). However, it is also estimated that the human genome

codes for about 26000-31000 proteins. There are a variety of proteomics techniques including

one-dimensional and two-dimensional gel electrophoresis (2-DE), as well as gel-free high-

throughput screening technologies such as multidimensional protein identification technology,

stable isotope labeling with amino acids in cell culture, isotope-coded affinity tag, and isobaric

tagging for relative and absolute quantitation. Shotgun proteomics, 2-Dimensional Difference

Gel Electrophoresis (2D-DIGE), and protein microarrays can be used in tissues, organelles,

and cells. Large-scale western blot assays, multiple reaction monitoring assays, and label-free

quantification of high mass resolution liquid chromatography (LC)-tandem mass spectrometry

(MS) are commonly used for high-throughput processing. In the last decade, proteomics has

been classified into protein expression mapping and protein interaction mapping. The former

method uses 2-DE combined with MS for quantitative proteome expression in cells, body

fluids, or tissues. Protein expression mapping can provide an understanding of the post-

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translational modifications of expressed proteins under different environmental conditions or

disease states. Protein-protein interaction mapping uses the yeast two-hybrid system coupled

with MS to determine the interaction partners for each cell’s encoded proteins and the

proteome-wide scale.

Proteomics is a multi-step technique in which every step should be very well controlled to

avoid non-biological factors interfering with protein expression and interaction. Sample

preparation is the most important step because it solubilizes all proteins in the sample and

eliminates all interfering inhibitory compounds such as lipids. Adequate sample preparation is

crucial to obtain reliable, accurate, and reproducible results. Polyacrylamide gel

electrophoresis (PAGE) is the most widely used method for protein separation and isolation.

High-performance Liquid Chromatography (HPLC), 1-Dimentional Electrophoresis, and 2-

Dimensional Electrophoresis are the methods used to separate proteins. Proteins are isolated

using 1- Dimensional Electrophoresis based on their molecular mass. Protein solubility is rarely

an issue since proteins are solubilized in Sodium dodecyl sulfate (SDS).

Furthermore, 1- Dimensional Electrophoresis is easy to use, repeatable, and capable of

resolving proteins with molecular masses ranging from 10 kDa to 300 kDa. As 1- Dimensional

Electrophoresis gel has minimal resolving power, it is most commonly used to characterize

proteins after being purified. However, in more complex protein mixtures, such as a crude cell

lysate, 2- Dimensional Electrophoresis may be used. In 2-DE, proteins are determined by their

net charge and their molecular mass.

Proteomics can analyze the expression of a protein at different levels allowing the assessment

of specific quantitative and qualitative cellular responses related to that protein. Qualitative and

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quantitative proteomes are measured at post-transcriptional, transcriptomic, and genomic

levels. According to the conditions, qualitative proteomics utilizes microarrays, 2-

Dimensional Electrophoresis, and 2D-LC to monitor protein mixture composition and protein

expression changes. In addition, it can provide information on the molecular mechanisms of

diseases and compare two groups such as patients with healthy controls. Quantitative

proteomics can also provide deep insights into disease mechanisms, cellular functions, and

biomarker discovery. Several new strategies are used in quantitative proteomics, such as post-

extraction or metabolic stable-isotope labeling alone or in combination with affinity labeling.

Mass Spectroscopy identifies compounds by sorting cations according to their mass-to-charge

ratio.

The study of proteomics has many applications in different fields such as medicine, oncology,

food microbiology, and agriculture. This report will shed light on proteomics, their techniques,

some of its applications, and the challenges currently faced in this field.

TYPES OF PROTEOMICS

Proteomics has three main types: expression proteomics, functional proteomics, and

structural proteomics.

 EXPRESSION PROTEOMICS

Expression proteomics is a novel approach that studies the quantitative and qualitative

expression of proteins. It aims to specify the difference in protein expression between two

conditions such as patients and controls. In addition, it can identify disease-specific proteins

and new proteins in signal transduction. Expression proteomics experiments are usually used

to study the patterns of protein expression in different cells. For example, a tumor tissue sample
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is compared to a normal tissue sample to identify differences in the levels of proteins.

Variations in protein expression, which are present or missing in tumor tissue compared to

normal tissue, are detected using 2-Dimensional Electrophoresis and Mass Spectroscopy

techniques.

 STRUCTURAL PROTEOMICS

Nuclear magnetic resonance spectroscopy and X-ray crystallography are used in structural

proteomics to determine the three-dimensional structure and structural complexities of

functional proteins. It specifies all protein interactions such as membranes, cell organelles, and

ribosomes in the mixture. The study of the nuclear pore complex is an example of structural

proteomics.

 FUNCTIONAL PROTEOMICS

This type of proteomics studies the protein functions and molecular mechanisms in the cell

and determines the protein partner’s interactions. In particular, it investigates the interaction of

an unknown protein with partners from a specific protein complex involved in a particular

process. This may indicate the biological role of the protein. In addition, the elucidation of

protein-protein interactions in vivo can lead to comprehensive descriptions of cellular signaling

pathways.

1.2 INTRODUCTION TO PERSONALIZED MEDICINE

Personalized medicine, also called precision medicine or individualized medicine, field

of medicine in which decisions concerning disease prevention, diagnosis, and treatment are

tailored to individual patients based on information derived from genetic and genomic data.

Personalized medicine centers on the concept that information about a patient’s genes and

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genome allows physicians to make more informed and effective decisions about a patient’s

care. This idea essentially is an extension of conventional medicine, in which one strategy is

applied across all patients, without tailoring to personal genetic and genomic information.

Personalized medicine is used in various ways to facilitate the prevention, diagnosis, and

treatment of disease. For example, physicians can use information on family history of disease

to assess a patient’s risk for a disease. In certain instances, family history can be used to

determine whether a patient should undergo genetic testing and, based on that information,

whether the individual would benefit from specific preventive measures. In the case of

individuals with a family history of Lynch syndrome (a cause of hereditary colorectal cancer),

for instance, detection of the causative mutation through genetic testing can be used to inform

decisions about screening. For persons who carry the mutation, frequent and routine screening

for evidence of precancerous lesions in the colon allows for early disease detection, which can

be a lifesaving measure. Similarly, tests capable of detecting mutations in multiple genes at

one time can assist in the early diagnosis of hereditary forms of breast cancer, ovarian cancer,

and prostate cancer.

1.3 RELATIONSHIP BETWEEN PROTEOMICS AND PERSONALIZED MEDICINE

Personalized medicine as a framework for disease diagnosis, treatment, and prevention at the

molecular level has entered clinical practice. From the start, genetics has been an indispensable

tool to understand and stratify the biology of chronic and complex diseases in precision

medicine. However, with the advances in biomedical and omics technologies, quantitative

proteomics is emerging as a powerful technology complementing genetics.

Personalized medicine aims to stratify patient populations so as to provide targeted and

efficient treatments and reduce adverse treatment effects for human health (König et al., 2017).

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Furthermore, it brings opportunities for the healthcare industry by utilizing novel diagnostics

platforms and specialized treatments that combine large-scale data with high-end

computational analysis (Flores et al., 2013; Siwy et al., 2019).

Proteomics is the next likely candidate to be included in the personalized medicine arsenal, for

proteins represent intermediate phenotypes. In particular, proteins are products of gene

expression and mediate biochemical activities of cells and tissues (Ding et al., 2019).

Proteomics approaches could describe disease-related pathways; identify novel biomarkers for

diagnostics; detect drug targets; and analyze physiological patterns on the transition for disease

(Van Eyk and Snyder, 2018).

More specifically, quantitative proteomics has emerged as an important technique for

personalized medicine because it provides information about the physiological differences

between biological samples based on the protein abundance levels. Thus, quantitative

proteomics has relevant applications for the clinical and biomedical field including biomarker

and drug discovery (Prasad et al., 2017). For the detection of human proteins, targeted

approaches are often used which include targeted mass spectrometry (MS) techniques or

affinity-reagent-based platforms. Targeted techniques aim to quantify the abundance of

preselected proteins from an individual and thus correlate concentration values with patterns

of disease.

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 CHAPTER TWO

PROTEOMICS TECHNIQUES

Mass spectrometry is an essential tool that is used for profiling proteins in the cell. However,

biomarker discovery remains the major challenge of proteomics because of their complexity

and dynamicity. Therefore, combining the proteomics approach with genomics and

bioinformatics will provide an understanding of the information of biological systems and their

disease alteration. However, most studies have investigated a small part of the proteins in the

blood. This review highlights the types of proteomics, the available proteomic techniques, and

their applications in different research fields.

2.1 PROTEOMICS WORKFLOW

Two methods can be used in proteomics: top-down and bottom-up workflows.

 BOTTOM-UP METHOD

The bottom-up method is sometimes called peptide-based proteomics. Here, the protein is

digested by trypsin and separated by a specific column, followed by analysis of the peptides

by Mass Spectrometer. The bottom-up approach can be classified into two groups according to

the fractionation step. The first approach uses 2-Dimesional Electrophoresis to isolate the

proteins from the gel. Then the proteins are digested into peptides that Mass Spectrometer can

identify. The second approach is called “shotgun” proteomics. Here, the digestion of protein

occurs without fractionation, and Liquid Chromatography is used to separate the peptides

identified by Mass Spectrometer.

 TOP-DOWN METHOD

In top-down proteomics, whole proteins or polypeptides are immediately assessed by

electrospray ionization (ESI) followed by matrix-assisted laser desorption/ionization (MALDI)

Mass Spectrometer. Top-down proteomics can identify proteins with a molecular mass of >

200 kDa.

Both approaches have various advantages and limitations. In the bottom-up approach, there is

low percentage coverage of the protein sequence, because the recovered sample includes small

and inconsistent fractions of total peptides. This results in missing a large proportion of

alternative splice variants and post-translational modifications. However, in top-down

proteomics, all characteristics of proteins are protected, and almost all existing modifications

and correlations can also be recovered. Moreover, in top-down proteomics, the

results of the exclusion of protein digestion with time are preserved. The major challenge in

top-down proteomics is the poor solubility of proteins compared to small peptides. Some

proteins in the membrane have high solubility but need to be washed with Sodium dodecyl

sulfate(SDS); however, SDS cannot be used in electrospray ionization. Proteomics workflows

involve sample preparation and analytical flow. The latter include separation of proteins,

protein identification, and validation.

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Figure 2.1 The differences between top-down and bottom-up proteomics. (Beeton-Kempen,

N. , 2020)

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SAMPLE PREPARATION

Proteomics experiments highly depend on the accuracy of sample preparation, in addition to a

well-designed pre-analytical workflow. There is no standard technique for sample preparation

in proteomics. Each method depends on the number of proteins in the sample, the sample’s

complexity, and the study’s objectives. Extraction of proteins from the mixture is the most vital

step in the preparation of samples. To maximize protein extraction and solubilization, the

extraction should include organic solvents and detergents followed by a tissue disruption

technique. The organic solvents and detergents can be removed by lyophilization. In previous

detergent-based methods, the extraction of 2,2,2-trifluoroethanol (TFE) macro-scale (> 100

µg) materials and nano-scale (30 µg)-based lysis have provided comparable protein detection

rate Basically, proteomics techniques involves two methods which are; Protein separation and

Protein identification.

2.2 SEPARATION AND ISOLATION OF PROTEIN

Gel-based and chromatography-based approaches are used for the separation and isolation of

proteins from the mixture.

2.2.1 GEL-BASED APPROACH

The best technique for protein isolation and detection is polyacrylamide gel electrophoresis.

For separation, 1-Dimensional Electrophoresis and 2-Dimensional Electrophoresis can be

used. Furthermore, 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) and Sodium

Dodecyl Sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) are examples of

2Dimensional variations used in gel electrophoresis.

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  1-DIMENSIONAL ELECTROPHORESIS (1-DE)

1-DE, can isolate proteins with a molecular weight of 10 kDa to 300 kDa. It uses SDS, a

detergent that denatures secondary and non-disulfide-linked tertiary structures, and combines

them with a negative charge proportional to their volume. This allows the calculation of

molecular weights. SDS-PAGE can be used to verify the purity of samples, test protein

purification, and calculate molecular weights for unknown proteins.

 2-DIMENSIONAL ELECTROPHORESIS (2-DE)

2-DE differentiates proteins better than 1-DE due to the variation in molecular weight and

isoelectric point of protein molecules. It also has a better resolution than 1-DE because the

protein is separated into two different dimensions. In 1-DE, the protein is separated based on

net charge, but in 2-DE, protein separation is based on the molecular mass and isoelectric point.

Thus, this method can detect different forms of proteins such as PTMs and phosphorylation.

Some proteins that arise from different proteolysis processes and splicing of alternative mRNA

can be resolved by 2-DE. There are many applications of 2-DE, including protein expression

profiling and cell map proteomics. Protein expression profiling can be used for comparing

normal and diseased tissues. Mapping proteins in 2-DE can be used in cellular organelles,

protein complexes, and microorganisms. 2-DE can help catalog proteins, and the database can

be created on the World Wide Web. However, 2-DE cannot detect proteins at a low molecular

weight and the limits of separation by isoelectric point and size.

2.2.2 CHROMATOGRAPHY-BASED APPROACH

Chromatography of affinity, size exclusion chromatography (SEC), and ion-exchange

chromatography (IEC) techniques can be used to purify protein-based chromatography. In

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addition, western blotting and the enzyme-linked immunosorbent assay are used to identify

selective proteins.

 ION EXCHANGE CHROMATOGRAPHY (IEC)

IEC is used to purify proteins according to their charges. This technique allows separating

proteins according to their charge nature, which is not possible by other approaches. The charge

accepted by the molecule of interest can be readily used by altering the pH of the buffer. The

IEC technique is low cost and can persist in variable buffer conditions.

 SIZE EXCLUSION CHROMATOGRAPHY (SEC)

SEC can be used to separate different compounds according to their size (hydrodynamic

volume) measured by how efficiently they enter the stationary phase’s pores. However, this

technique is not as useful as other proteomics techniques. Two basic versions of SEC are

utilized: gel permeation chromatography (GPC) using organic solvents, which is used for

polymer analysis; and gel filtration, which is performed using aqueous solvents.

 LIQUID CHROMATOGRAPHY (LC)

Liquid Chromatography is a powerful technique that can separate proteins from a complex

mixture and can analyze large and fragile biomolecules. When combined with MS, it can be

used for determining the peptides in the mixture. Liquid Chromatography can help researchers

discover novel biomarkers and understand the mechanisms of carcinogenesis according to the

modification of proteins. For example, some researchers use LC-MS to rapidly monitor

congenital adrenal hyperplasia from dried filter-paper blood samples.

2.3 PROTEIN IDENTIFICATION AND CHARACTERIZATION

The identification of proteins is a critical step in proteomics. Mass Spectrometer can be used
12
after the separation of the proteins by chromatography or electrophoresis. Other techniques can

also identify proteins such as Edman sequencing and protein microarray.

2.3.1 MASS SPECTROMETER (MS)

Mass Spectrometer is the best analytical tool for rapidly facilitating the sequencing of proteins.

It can also be used to detect the molecular weight of proteins. In this technique, protein

molecules are ionized, and their mass is calculated according to mass-to-charge ratios. The

mass spectrometer has three main components: an analyzer, an ion source, and a detector. The

methods used for ionization are electrospray ionization (ESI) and matrix-assisted laser

desorption/ionization (MALDI).

 MATRIX-ASSISTED LASER DESORPTION/IONIZATION

In matrix-assisted laser desorption/ionization, a chemical matrix is mixed with the peptides,

and spotted onto a metal multiwall microliter plate to make a crystal lattice. The matrix

chemicals pass the energy to the samples after absorbing it. Then peptide ions are detected by

a mass analyzer. Matrix-assisted laser desorption/ionization creates mostly singly charged ions

that help to determine the m/z value.

 ELECTROSPRAY IONIZATION

In electrospray ionization, the power is activated in the protein sample to create charged

droplets that increase gaseous ion production, which then are analyzed with a mass analyzer.

The advantages of electrospray ionization are its high reproducibility and high elasticity to

combine many categories of Mass spectrometer. Furthermore, electrospray ionization can be

fixed to time-of-flight (TOF)-MS, quadruple, ion traps, and fourier transform ion cyclotron

resonance. On the other hand, the disadvantages of electrospray ionization are that it cannot be

13
applied for molecular imaging, it requires a large quantity of samples, and multiple peaks are

produced due to the many charged ions that result in the complexity of MS/MS spectra.

14
Figure 2.2 Matrix-Assisted Laser Desorption/Ionization and Electrospray Ionization

(Beeton-Kempen, N. et al., 2020)

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2.3.2 EDMAN SEQUENCING

Edman sequencing has been used to detect the sequence of amino acids in peptides or proteins.

This technique includes the reaction of chemicals, which remove and determine amino acid

residues present at the N-terminus of the polypeptide chain. Thus, it plays a significant role in

assessing biopharmaceutical quality and therapeutic proteins.

2.3.3 PROTEIN MICROARRAY

Protein microarrays are used to determine the function of protein, as well as to monitor their

interactions. The structure of the array allows for numerous proteins to be tracked in parallel.

Protein microarrays were developed by utilizing the technology of DNA microarrays, which

are commonly used to analyze gene expression. The requirements of protein microarrays are,

however, more complex and necessitate material customization to make them suitable. Protein

microarrays apply small amounts of sample to a “chip” for analysis (this is sometimes in the

form of a glass slide with a chemically modified surface). Specific antibodies can be

immobilized to the chip surface and used to capture target proteins in a complex sample. This

is termed an analytical protein microarray, and these types of microarray are used to measure

the expression levels and binding affinities of proteins in a sample. Functional protein

microarrays are used to characterize protein functions such as protein–RNA interactions and

enzyme-substrate turnover. In a reverse-phase protein microarray, proteins from e.g., healthy

vs. diseased tissues or untreated vs. treated cells are bound to the chip, and the chip is then

probed with antibodies against the target proteins.

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Figure 2.3 Protein microarray (Beeton-Kempen, N. , 2020)

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2.4 PROTEIN IDENTIFICATION AND VALIDATION

Sequent, Mascot, Comet, and Tandem are instruments currently available for database

searching. However, most search devices do not produce matching data as they operate on

differentiation algorithms and recording functions, creation integration, and data comparison

from many studies and experiments. As a result, the identification of peptides by data search

needs additional time. High-quality data makes the data search more effective and less time

consuming. Moreover, using accurate mass to measure ion fragments can shorten database

explorations and produce more accurate results.

 WHAT TANDEM IN MS?

A tandem mass spectrometry (TANDEM MS), also named as MS/MS, is a two-step technique

used to analyze a sample either by using two or more mass spectrometers connected to each

other or a single mass spectrometer by several analyzers arranged one after another.

Peptides can be subjected to multiple rounds of fragmentation and mass analysis—a process

termed tandem-MS, Mass spectrometer by combining the same or different mass analyzers in

tandem, such as Quadrupole-Time Of Flight (Q-TOF) or triple-quadrupole (QQQ) Mass

Spectrometer, the strengths of different mass analyzers can be leveraged to further improve the

capacity for proteome-wide analysis. Simple MS setups such as Matrix-assisted laser

desorption/ionization-Time-Of-Flight are used for peptide mass measurements, whereas

tandem mass spectrometers are used to determine peptide sequences.

2.5 BIOINFORMATICS IN PROTEOMICS

Bioinformatics analyses use novel proteomics algorithms to manage the large and varied data

in the process of marker discovery. Controlling this massive quantity of data and finding the

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association between other omics technologies (e.g., metabolomics and genomics) remain

difficult. The analysis of proteomics data is challenging because of the parameters used in

processing, quality valuation, and shortage of standards for data formats. The big challenge is

how to analyze massive data and create real biological understanding. Protein pathways are a

collection of internal cell reactions that have a specific biological impact.

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 CHAPTER THREE

APPLICATION OF PROTEOMICS

Personalized medicine involves the eradication of “one-fit-all” mode of diagnosis which entails

the study of the protein content of an individual before treatment. Proteomics is a revolutionary

technique that has been used in medicine, including drug and biomarker discovery. Proteomics

can identify and monitor biomarkers by analyzing the proteins in the body fluids such as urine,

serum, exhaled breath and spinal fluid. Proteomics can also facilitate drug development by

providing a comprehensive map of protein interactions associated with disease pathways.

3.1 BIOMARKER DISCOVERY

The term “biomarker”, a portmanteau of “biological marker”, refers to a broad subcategory of

medical signs – that is, objective indications of medical state observed from outside the patient

which can be measured accurately and reproducibly. In molecular terms biomarker is "the

subset of markers that might be discovered using genomics, proteomics technologies or

imaging technologies. Biomarkers play major roles in medicinal biology. Biomarkers help in

early diagnosis, disease prevention, drug target identification, drug response etc.

A biomarker is an assessable pointer of a normal or abnormal biological state in the body. In

clinical settings, cancer development and its response to therapy are measured by cancer

biomarkers. 2D-PAGE is used for the discovery of biomarkers. It can also compare the profiles

of proteins in normal and diseased cells such as tumor tissues and body fluids. Cancer

biomarkers are divided into three classes, predictive, prognostic and diagnostic, based on their

uses. Predictive biomarkers can predict the response to therapy. For instance, in breast cancer,

the activation and the positivity of human epidermal growth factor receptor 2 can predict the

response to trastuzumab. In addition, in colorectal cancer, mutation of Kirsten rat sarcoma virus
20
gene can predict resistance to treatment with epidermal growth factor receptor inhibitors (e.g.,

cetuximab).

On the other hand, prognostic biomarkers can provide physicians with a prediction of the

clinical outcomes. For example, the 21-gene repetition mark predicts breast cancer relapse and

complete survival in node-negative, tamoxifen-treated breast cancer. The third group of

biomarkers is the diagnostic biomarker, which indicates if a patient has a specific disease

condition. For example, in colorectal cancer, a stool DNA test is used as a diagnostic

biomarker. These biomarkers can be found in tissues, serum, blood, and urine. The body-fluid

sampling for proteomics is thus less invasive and low cost. The discovery of biomarkers has

progressed in many diseases such as acquired immune deficiency syndrome, cardiovascular

diseases, diabetes, cancer, and renal diseases. However, the highly complex mixtures of

proteins and the high range of protein dynamics are examples of challenges in fluid sampling

for proteomics. Each type of sample has a different usage according to the disease conditions.

For instance, in kidney disease, the urine sample is used to assess urine proteins, reflecting

changes in kidney functions. In other human diseases, blood is also used for biomarker

discovery. There are some challenges for using the plasma in biomarker discovery, such as

protein dynamicity, the variation of the patient, and the low abundance of biomarkers in

plasma. These challenges in biomarker discovery have yet to be addressed. Most biomarker

discovery studies are focused on cancer-related diseases due to their clinical importance. For

instance, many biomarkers are associated with tumors that can be used to follow up with the

patients.

Much of the current research directing the use of proteomics in the clinic or hospital is for the

identification of biomarkers, though it largely remains in the discovery phase. The search for

biomarkers can largely be broken down into two categories: those specific for the diagnosis of
21
illness and those associated with disease severity. Ongoing research has already identified

biomarkers of varying reliability for numerous cancers, intestinal bowel disease, amyotrophic

lateral sclerosis, and other diseases.

Methodologies to quantitate biomarkers other than by mass spectrometry have demonstrated

great success in the clinic. However, the limitations of these alternative strategies are namely

two-fold: the number of analytes and its sensitivity. Current clinical assays to quantitate

biomarkers generally analyze only a single molecule at a time. Complicated diagnoses may

require analysis of multiple biomarkers to either rule in or rule out various diseases, taking

precious time. In contrast, analysis of biomarkers using mass spectrometry can quantitate

numerous biomarkers from a single sample and in a single test. Multiplexing the analysis of a

patient sample could significantly reduce the time required for a patient’s diagnosis. Several

biomarkers detectable by mass spectrometry have already been associated with cardiac events

(trimethylamine-N-oxide, MMP-2 MMP-9, MMP10, NGAL), intestinal bowel disease, and

numerous targets for various cancers.

Most known biomarkers come from proteins present at high levels in the serum or tissue, e.g.,

albumin, CRP, OVA1, etc. The challenge for precision medicine is to develop new biomarkers

making use of the exquisite sensitivity of mass spectrometry for detection of biomarkers at

lower abundance. These biomarkers may not necessarily be levels of protein expression but

rather may include posttranslational modifications (PTMs), metabolites, or metabolic flux. In

addition, identifying the appropriate molecular isoform from a complex mixture is necessary

for several existing biomarkers. The sensitivity of mass spectrometry in the detection of

addition of a phosphate, fucosyl group, ubiquitin, or glycosylation sumoylation, etc. makes

mass spectrometry ideal for determining the relative abundance of these isoforms in disease

association.
22
Many proteins exist in multiple states, either post-translationally modified or enzymatically

cleaved. These PTMs and cleavages can be responsible for activation of the protein resulting

in the disease state, or removal/inactivation of the protein causing the same. Therefore, the

relative abundance of the precursor and processed forms of these proteins indicate disease or

the predisposition for developing disease. For example, bioactive insulin is synthesized as

preproinsulin containing a secretory signal peptide and an extraneous intramolecular fragment,

C-peptide. Following removal of the signal peptides, proinsulin is packaged into secretory

vesicles and the C-peptide is enzymatically excised from the interior of the proinsulin. Thus,

all three forms of the protein contain the sequence of bioactive insulin. Since the precursor and

processed proteins share much sequence, and even structural, identity, determining the ratio of

each can be difficult with immunoassays. However, proteomic technology is designed to

determine the relative loss or gain of mass units. In addition to insulin, growth hormone,

parathyroid hormone, and albumin all exist as a heterogeneous mixture of various protein states

with their relative abundances of potential clinical import. Moreover, the aberrant production

of a single clone of antibody is associated with several diseases, e.g., osteomyelitis,

macroglobulinemia, and multiple myeloma. Tandem mass spectrometric sequencing of

immunoglobulin chains can identify the overproduction of a given monoclonal antibody

specificity amongst the numerous antibody specificities present at any given time in patient

serum.

Furthermore, analysis of the proteomes of clinical specimens (tissues, bodily fluids, cultured

primary cells) associated with a specific condition/illness can lead to the identification of novel

biomarkers. Relative differences in the proteomes give an unbiased comparison between

normal and affected samples. This untargeted approach to the discovery of novel biomarkers

eliminates the need for a priori research or a systematic understanding of the condition before

23
a biomarker can be identified. This untargeted, unbiased approach is particularly useful for

new, rare or neglected diseases where little is understood about the physiology of the disease

or the causative agent.

Another area where proteomics and mass spectrometry excel is the analysis of small molecule

metabolites. The presence and/or change of metabolite concentrations (metabolic flux) can

signify an alteration in the normal functioning of a patient’s physiological state. Alterations in

energy consumption and the ‘oncometabolite’ R-2-hydroxyglutarate have both been linked to

cancer. In theory, changes in a patient’s metabolites detected by mass spectrometry could

indicate the presence of a tumor in a remote location not easily detectable by conventional

means. Based on the specific metabolic flux, the treatment could therefore be adapted to the

individual patient’s condition. However, this method of cancer diagnosis remains to be

confirmed in a clinical setting.

3.2 DRUG DISCOVERY AND DEVELOPMENT

Drug discovery is a complex process with many different stages including chemical, functional,

and clinical proteomics-based approaches. The application of proteomics in drug discovery has

been developed to include patients’ treatment and care. 2-DE cannot be used in drug discovery

because it fails to separate the membrane proteins that characterized about 50% of important

drug targets. Moreover, 2-DE cannot detect low-abundance proteins. In drug discovery

proteomics, understanding the function of proteins and their interactions in the mixture is very

important. Also, the methods should be able to detect low-abundance proteins and their

activity. Therefore, many technologies such as Mass Spectroscopy and protein-chip have been

used to identify and separate phage proteins. In addition, other techniques such as activity-

based assays and two-hybrid assays can be used for the same purpose. Using 2D-PAGE-

24
MALDI-TOF/TOF, Lavandula angustifolia was used as a drug to treat Alzheimer’s disease in

rats.

3.3 ONCOLOGY

The application of proteomics in cancer is called oncoproteomics. Oncoproteomics can be used

to identify anticancer drugs and the personalization of cancer management. Microarrays and

laser capture microdissection (LCM) of the tumor tissue can classify proteins in cancer.

Oncoproteomics applications are used in many tissues such as the colon, breast, rectum,

prostate, and brain. In addition, proteomics can be used to diagnose cancer and discover novel

therapies. Many proteomics techniques can be used to detect biomarkers in cancer such as

aptamer-based molecular probes, cancer immunomics, tissue microarrays, nano-proteomics (to

isolate signatures of autoantibodies), and antibody microarrays.

Analytical platforms for proteomics have been developed to identify the entire set of proteins

in organisms and to uncover qualitative and quantitative protein variations upon diverse

environmental changes. In addition, comprehensive research on proteins has become possible

by building an amino acid sequence database on the composition of proteins. Generally, a

proteomic analysis consists of the following steps:

1. protein extraction,

2. protease digestion,

3. peptide fractionation,

4. LC-MS analysis (Fig. 3.1).

Initially, proteins are extracted and purified from tissue or cell lysates by centrifugation and

filtration. Then, the protein mixture is typically separated by two-dimensional gel

analysis of their peptides, which are produced by enzymatic (usually tryptic) digestion, and the

data are interpreted using a proteome database.

26
Figure 3.1 Steps involved in proteomics analysis (Kwon, Y. et al. 2021)

27
An attractive part of the proteomics field is its ability to reveal novel biomarkers of diseases.

For example, as cancer progresses, changes in protein profiles and differences in protein

distribution both in tissues and body fluids such as blood can be examined through quantitative

analysis. Proteomics enable the simultaneous qualitative and quantitative profiling of

numerous proteins. Liquid chromatography-mass spectrometry (LC/MS) is a key technique

that obtains high-resolution spectra of mixed peptides, allowing the discovery of sensitive and

specific biomarkers associated with cancer. The high-throughput technologies based on this

technique enable semi-quantitative and quantitative analyses. For the quantitative study of

proteins, label-based and label-free approaches are widely used in clinical research(Table 3.1).

Label-based quantitation strategies allow the quantitative and qualitative analysis of proteins

in a sample. The methods consist in using stable isotope labeling of compound markers such

as amino acids to tag proteins or peptides. The samples containing the tagged proteins are then

compared with control proteins tagged with isotope-free markers. These methods have the

advantage of minimizing disparities between individually handled samples. However, proteins

may be partially labeled and the reagents are expensive. In proteomics, the common labeling

methods are SILAC, ICAT, TMT, and iTRAC. SILAC has the least experimental variability

because it is a metabolic labeling method; the isotope reagent is used in the initial step of

sample preparation (i.e., cell culture) and the labeled proteins are generated through metabolic

processes. ICAT is a chemical labeling technique that uses a reagent consisting of a functional

group that targets cysteinyl residues, a deuterium atom-based linker region, and a biotin group

for protein purification. Sample complexity is significantly reduced through affinity-based

extraction of labeled proteins. Isobaric labeling methods using TMT and iTRAC need tandem

MS techniques. Labeling reagents containing reporter ions are produced under tandem MS,

and their amount is proportional to that of tagged peptides, resulting in the quantitation of

28
proteins. In contrast to label-based strategies, label-free quantitation approaches using MRM

or SWATH are straightforward without labeling steps, which is suitable for large-scale studies.

Label-free proteins are quantified based on the signal intensities or spectral counts of peptides

unique to them, which are obtained from the MS analysis. The recent development of high-

resolution mass spectrometers has led to advances in label-free quantitation for proteomics.

Label-free quantitation is easy to use, yields highly reproducible results in biochemical

experiments, and is reliable when many statistical verifications are required. The amount of

protein can also be analyzed using antibody arrays such as the ELISA. This is a semi-

quantitative and quantitative analysis in which capture antibodies are immobilized on a solid

surface such as a nitrocellulose membrane, glass slide, silicone, or beads. Then, the interaction

between the antibody and its target protein is detected. However, it is not a discovery-oriented

method, and it is limited to the detection of usable and compatible proteins.

29
Table 3.1: Table showing different quantification methods with their advantages and

disadvantages (Kwon, Y. et al,. 2021).

30
3.4 PERSONALIZED PROTEOMICS

These advances in high throughput mass spectrometry of clinical samples allowed for the

publication of a draft of the human proteome. This collection of data from various tissues of

healthy samples forms the basis for comparative clinical proteomics. By comparison of patient

samples with this database, scientists associate changes in the proteomes with particular disease

states. Translation of these findings into the clinic and hospital setting allows doctors the

possibility of mass spectral analysis of patient proteomes. While initial comparison must be

made between the patient sample and the database average, as bioinformatics and information

processing grows, ultimately physicians will be able to compare an ill patient’s proteome with

their own healthy archived records. This Personalized Proteomic analysis has the potential to

greatly increase the diagnostic accuracy while reducing time and costs.

Through rapid proteomic screening of easily accessible tissue samples, early determination of

the presence and/or severity of disease allows for quick response from medical personnel and

improved patient outcome. While this is certainly useful for biomarker monitoring, it places

strong technological and monetary constraints on a burgeoning medical field. The power of

proteomics is in discovering the meaningful unknown in a bouillabaisse of unrelated molecules

with high mass accuracy and sensitivity. For this reason, clinical proteomics will be most useful

for diagnosis of diseases with rare or unknown etiology, monitoring the therapeutic effect and

activity of drug regimens, improving treatment options for individual patients, and, as always,

discovering a brave new world of medical possibilities.

3.4.1 PROTEOMICS IN DIAGNOSIS

Rapid identification of an infectious disease remains challenging and time consuming often

requiring multiple tests and even days of culture. Assemblage of databases containing the

31
proteomes of humans and their pathogens for comparison to samples obtained in the clinic can

result in immediate diagnosis of an infection. Already, proteomic analysis can identify E.

coli, Salmonella, Campylobacter, Clostridium (including C.difficile), L.monocytogenes, Myco

bacterium, Staphylococci, H. pylori, and enterobacteriaceae from biological samples. Many of

these species contain multiple strains each encoding its individual combination of toxins and/or

lethal factors. Isolate testing by proteomics has been reported to be faster and less costly for

many of these infections. Moreover, a single test can determine not only the bacterial strain but

also antibiotic resistance mechanisms reliant upon alterations in protein expression.

Viral infections can be misdiagnosed as bacterial infections due to the use of clinical tests

detecting levels of C-Reactive Protein (CRP) and IL-6 production, both of which are similarly

upregulated in response to bacterial and viral infections, leading to prescription of ineffectual

antibiotics for viral infection. Rather, proteomic analysis of a patient sample can reveal both

unique host proteins as well as specifically bacterial or viral proteins resulting in a more

accurate diagnosis and treatment. In addition, the success of a given therapeutic plan varies

among patients being more or less effective for particular subpopulations of patients and for

different stages of disease. Therefore, monitoring the efficacy of antibiotics or antivirals after

administration could quickly detect resistance or ineffectiveness of a particular treatment.

In addition, monitoring the processing, activation, and clearance of therapeutics could provide

physicians with real time functional information about the success of the treatments

administered (in clinica pharmacodynamics). Proteomic analysis of serial patient samples

could potentially detect activation or breakdown of a therapeutic agent allowing physicians to

monitor the therapeutic dose. Proteomic-based pharmacokinetic measurements of bio-

therapeutics have already been shown to be suitable alternatives to traditional assays. Serial

proteomic analysis could also monitor the progress, clearance, or lack thereof of a disease, e.g.,
32
infection or cancer, for each individual patient. As tumors are forced into remission, the altered

metabolite profile discussed above should return to normal; conversely, proteomic analysis of

tumors unresponsive to treatment would show no change. In addition, it was shown that the

host peptidome, and likely the proteome from which these peptides were derived, changes in

response to viral infection. Therefore, even in autonomous transplant recipients, an infection

in the host after transplant could alter the peptide repertoire presented by the transplant leading

to rejection. Mass spectrometric monitoring for changes in the host peptidome following

transplant could detect the potential for such viral-mediated peptidome shift and rejection,

allowing for early intervention to tolerate the host to the new peptidome.

The detection of mass and mass losses by mass spectrometry also provides strong advantages

for in clinica measurements of protein kinetics. This could be the activation of proteins through

posttranslational modifications, as discussed above, but also rate of protein synthesis and

clearance. Changes in these rates affect the overall concentration of target proteins, both host

and/or biotherapeutic, which can inform dosing strategies. The high sensitivity and mass

accuracy of the mass spectrometer makes it ideal for measurements of changes in these often

low abundance proteins.

3.4.2 PROTEOMICS TO IMPROVE PATIENT/TREATMENT SELECTION

The goal of precision medicine is to more specifically and reliably match the patient with the

best course of therapy to ensure the optimal outcome for each individual patient. Just as blood

group typing reduced complications with transfusions, genetic profiling of HLA loci has

reduced transplant rejection rates by matching donor and recipient. Despite this level of

scrutiny, as many as 10% of transplants result in rejection or graft versus host disease (GVHD).

Differential alteration of the self and transplant peptidome after infection has been implicated

33
as a potential cause for these complications. Personalized proteomic analysis of the HLA loci,

minor antigen loci, and peptide repertoire of potential donor/recipient pairs could reduce this

rate even further. Analysis at this level of specificity and detail requires the accuracy and

sensitivity of mass spectrometry.

34
 CHAPTER FOUR

CONCLUSION

There are several difficulties in the study of proteins that are not inherent in the study of nucleic

acids. Proteins are more difficult to work with than DNA and RNA. They have secondary and

tertiary structure that must often be maintained during their analysis. Proteins can be denatured

by the action of enzymes, heat, light, or by aggressive mixing as in beating egg whites. Some

proteins are difficult to analyze due to their poor solubility.

On the other hand, proteins cannot be amplified like DNA, therefore less abundant species are

more difficult to detect. Approximately half of the total protein content in plasma comes from

albumin (∽55 mg/mL), and together with another 10 proteins, they make up 90% of the total

content. Low abundant proteins such as cytokines are normally present at 1–5 pg/mL. Each

proteomics technology can only analyze proteins within 3–4 orders of magnitude, and mostly

at the higher concentration end of the spectrum. The removal of high abundant proteins from

plasma or serum is thus a prerequisite for conducting more detailed proteomics studies on low

abundant proteins. However, many potentially important biomarkers may be lost in this process

due to non-specific binding or the co-removal of proteins/peptides intrinsically bound to the

high abundant carrier proteins. In particular albumin, removal has been shown to result in

significant loss of cytokines. Thulasiraman et al. developed the new deep proteome approach

via ligand library beads. The use of equalizer beads coupled with a combinatorial library of

ligands has been shown to allow access of many low abundant proteins or polypeptides

undetectable by classical analytical methods. The population of beads has such diversity that a

binding partner should exist for most proteins in a sample. Each bead has an equivalent binding

capacity. High abundant proteins saturate their binding partners and the excess proteins are

35
washed away, whereas trace proteins are concentrated on their specific affinity ligands. This

panoply of methods could offer a strong step forward in “mining below the tip of the iceberg”

for detecting the “unseen proteome”.

To tackle with the sensitivity issue, constructed the ultramicroarrays, combining the advantages

of microarray including multiplexing capabilities, higher throughput, and cost savings with the

ability to screen very small sample volumes. These ultramicroarrays were found to have a high

specificity and sensitivity with detection levels using purified proteins in the attomole range.

This strategy enables the proteomics analysis of materials that are available in very limited

quantities, such as those collected by laser capture microdissection, neonatal biopsy

microspecimens, and forensic samples.

Apparently, a number of technical obstacles remain before routine proteomics analysis can be

achieved in the clinic. However, the standardization of methodologies and dissemination of

proteomics data into publicly available databases is starting to overcome these hurdles. The

cost is also a precluding factor for the widespread use of proteomics in clinical laboratory. Most

proteomics technologies use complex instrumentation, critical computing power, and

expensive consumables. Another major challenge will be the integration of proteomics with

genomics and metabolomics data, as well as their functional interpretation in conjunction with

clinical results and epidemiology.

4.1 THE FUTURE OF PROTEOMICS

Currently, proteomic workflows rely heavily on MS. As powerful as this technology has

proven, researchers are now looking ahead to a future for proteomics that lies “beyond MS.”

Despite the attomolar sensitivity of MS, millions of the target molecule still need to be present

in the sample for it to be detected. This implies that low-concentration target molecules (e.g.,

36
serum biomarkers) can be undetectable in complex milieu such as human serum unless first

enriched for.

Scientists are still searching for the holy grail of high-throughput proteomic techniques that

1) Has excellent sensitivity across the dynamic range of the target proteome (e.g., 10 for the

human proteome),

2) Can directly read entire protein sequences and identify their PTMs, and, therefore

3) Does not need to draw inferences from databases of theoretical protein matches.

There are several promising technologies that, while currently hampered by limitations in

sensitivity, throughput or cost, may yet come to dominate the proteomic field. These include

nascent fluorescent fingerprinting methods and yet-to-be-developed subnanopore arrays for

the high-throughput single-molecule sequencing of proteins.

Along with the expected advances in proteomic techniques, approaches to proteomic data

analysis are expected to evolve just as rapidly. For example, there is a strong impetus towards

developing data technologies such as cloud computing, software containers and workflow

systems, which will “democratize” access to top-notch computing resources for proteomic data

analysis regardless of researchers’ location, IT infrastructure or computational expertise.

The use of proteomics in the clinic is the future of personalized medicine. However, the utility

of personalized proteomics for diagnosis and treatment is dependent upon the meaningful

translation of basic science research to medicine. The power of proteomics for use in clinica is

also that which proves problematic for basic research. The translatability of basic research to

the clinic has so far been limited due to the failure of biomarkers to apply to a large population.

More stringency during the discovery phase with better verification of biomarkers and

37
validation in preclinical studies are needed. This is evident in the use of single cell proteomics

in basic and applied research despite its limited utility in clinica as most conditions for which

a patient might seek treatment involve the interaction of multiple cells and cell types and not

the dysfunction of a single cell. Just because it is possible to generate a large amount of

hypersensitive data mass accurate to four decimal places from a tiny amount of starting material

does not necessarily mean this is useful to clinicians. Integration of basic and clinical research

is needed to develop biomarkers and assays to definitively identify differential proteomics

between healthy and diseased patient samples.

38
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41
 APPENDIX

2D-PAGE - 2 Dimensional Polyacrylamide Gel Electrophoresis

CPR - C-Reactive Protein

D-DIGE - Dimensional Difference Gel Electrophoresis

DE - Dimensional Electrophoresis

D-LC - Dimensional Liquid Chromatography

DNA - Deoxyribonucleic Acid

ELISA - Enzyme-Linked Immunosorbent Assay

ESI - Electrospray Ionization

GVHD - Graft Versus Host Disease

HLA - Human Leukocyte Antigen

HPLC - High-performance Liquid Chromatography

ICAT - Individualized Cancer Therapy

IEC - Ion-Exchange Chromatography

IL-6 - Interlukin 6

iTRAC - Translational Research Acceleration Collaboration

LC - Liquid Chromatography

LCM - Laser Capture Microdissection

MALDI - Matrix-Assisted Laser Desorption/Ionization

42
MS - Mass Spectrometry

PAGE - Polyacrylamide Gel Electrophoresis

PTMs - Post-Translational Modifications

Q-TOF - Quadrupole-Time Of Flight

RNA - Ribonucleic Acid

SDS - Sodium Dodecyl Sulfate

SDS-PAGE - Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis

SEC - Size Exclusion Chromatography

SILAC - Stable Isotope Labeling

TMT - Trimodality Therapy Treatment

TOF - Time-Of-Flight

43