# Proteomics/Introduction to Proteomics

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## What is proteomics?

!Information transfer in the central dogma of
biology
The focus of proteomics is a biological group called the proteome. The
proteome is dynamic, defined as the set of
proteins expressed in a specific cell, given a particular set of
conditions. Within a given human proteome, the number of proteins can be
as large as 2 million.

Proteins themselves are macromolecules: long chains of amino
acids. This amino acid chain is constructed
when the cellular machinery of the ribosome
translates RNA transcripts from DNA in the cell\'s nucleus.
 The transfer of information within cells
commonly follows this path, from DNA to
RNA to protein.

Proteins can be organized in four structural levels:

:\*Primary (1°): The amino acid
sequence, containing members of a (usually) twenty-unit alphabet

:\*Secondary (2°): Local folding of
the amino acid sequence into α helices and β sheets

:\*Tertiary (3°): 3D conformation of
the entire amino acid sequence

:\*Quaternary (4°): Interaction
between multiple small peptides or protein subunits to create a large
unit Each level of protein structure is essential to the finished
molecule\'s function. The primary sequence of the amino acid chain
determines where secondary structures will form, as well as the overall
shape of the final 3D conformation. The 3D conformation of each small
peptide or subunit determines the final
structure and function of a protein conglomerate.

There are many different subdivisions of proteomics, including:

:\*Structural proteomics \--
in-depth analysis of protein structure

:\*Expression proteomics \--
analysis of expression and differential expression of proteins

:\*Interaction proteomics \--
analysis of interactions between proteins to characterize complexes and
determine function.

Proteomics has both a physical laboratory component and a computational
component. These two parts are often linked together; at times data
derived from laboratory work can be fed directly into sequence and
structure prediction
algorithms. Mass spectrometry of
multiple types is used most frequently for this purpose.

## The importance of proteomics

Proteomics is a relatively recent field; the term was coined in 1994,
and the science itself had its origins in electrophoretic separation
techniques#Molecular_Biology_and_Biochemistry "wikilink")
of the 1970\'s and 1980\'s.  The study of
proteins, however, has been a scientific focus for a much longer time.
Studying proteins generates insight on how proteins affect cell
processes. Conversely, this study also investigates how proteins
themselves are affected by cell processes or the external environment.

Proteins provide intricate control of cellular machinery, and are in
many cases components of that same machinery.
 They serve a variety of functions within
the cell, and there are thousands of distinct proteins and peptides in
almost every organism. This great variety comes from a phenomenon known
as alternative splicing, in which a
particular gene in a cell\'s DNA can create multiple protein types,
based on the demands of the cell at a given time.

The goal of proteomics is to analyze the varying proteomes of an
organism at different times, in order to highlight differences between
them. Put more simply, proteomics analyzes the structure and function of
biological systems.  For example, the protein
content of a cancerous cell is often different from that of a healthy
cell. Certain proteins in the cancerous cell may not be present in the
healthy cell, making these unique proteins good
targets for anti-cancer drugs. The realization
of this goal is difficult; both purification and identification of
proteins in any organism can be hindered by a multitude of biological
and environmental factors.

!Protein structural levels of interest in
proteomics

## Proteomics Workflows

The first step of proteomics is sample preparation. In this step, we are
trying to extract protein from cells. In the second step, we use methods
such as 2D
electrophoresis to
separate different proteins. Then we try to cut proteins into peptides
since peptides are easier to detect. In the forth step, we use mass
spectrometry to detect peptides and peptides fragments. Finally, we can
then determine the sequence of the protein by interpreting all the data
obtained.

![](targeted_mass_spectrometry.gif "targeted_mass_spectrometry.gif"){width="400"
height="400"}

## Broad-Based Proteomics

!Broad-based Proteomics Approach vs traditional focused
approach{width="600"}
Because Proteomics is growing at a very rapid pace, there is a shift in
the field away from a specialized/focused way of conducting studies and
towards a more global perspective. Broad-based proteomics presents a
unique perspective on the field of proteomics because it allows for one
to take on this general perspective by setting out to understand the
proteome as a whole. A critical aspect to this strategy is planning
ahead; and in doing so, the most appropriate plans and technologies can
be implemented in the most efficient manner. By developing a strategy
tailored to understanding a particular proteome, problems and setbacks
can be avoided during the study.

The first step when utilizing broad-based proteomics is to develop a
hypothesis specific to the proteome being studied. It is best to choose
organisms that already have a great deal of
genomic information
available, since the genome is always a useful supplement to proteomic
information. Once the a hypothesis and organism are established, the
proper technologies should be chosen; and these technologies should be
compatible with whatever biological factors are present (i.e. sample
type). Some important and relevant proteomic methods include
HPLC, Mass
Spectrometry,
SDS-PAGE, two-dimensional gel
electrophoresis, and
perhaps in silico protein
modeling.

Since there are multitudes of sample type, sample preparation, and
analytical technology combinations possible, it is obvious why careful
planning from a broad-based proteomic perspective is critical. By
planning upfront, an efficient proteomic study can be conducted. And
when the efforts of many broad-based proteomic studies are taken
together, understanding the proteome in its entirety becomes a realistic
possibility.

## References

1.      American Medical Association. \"Proteomics.\"
    <http://www.ama-assn.org/ama/pub/category/3668.html>

2.      Hartl, Daniel L., Jones, Elizabeth W. \"Genetics: Analysis of Genes
    and Genomes\". Jones and Bartlett Publishers: Boston, 2005.

3.      Weaver, Robert F. \"Molecular Biology, 2nd Edition\". McGraw Hill:
    Boston, 2002.

4.      Twyman, Richard. \"Proteomics.\"
    <http://genome.wellcome.ac.uk/doc_wtd020767.html>

5.      Colinge, Jacques and Keiryn L. Bennett. \"Introduction to
    Computational Proteomics\". PLoS Comput Biol. 2007 July; 3(7): e114.

6.      \"History of Proteomics.\" Australian Proteome Analysis Facility.
    <http://www.proteome.org.au/History-of-Proteomics/default.aspx>

7.      Graves, P. R., T. A. J. Haystead. \"Molecular Biologist\'s Guide to
    Proteomics\". Microbiology and Molecular Biology Reviews: Vol.66
    No.1, 2002.

8.      \"Proteomics Overview.\" <http://www.proteomicworld.org/>

9.      van Wijk, K. J. \"Challenges and Prospects of Plant Proteomics\".
    Plant Physiol. 2001 June; 126(2): 501-508.

Chapter Written by J. Reuter (Zel2008) and S. Lafergola (DieselSandwich)

## Articles Summarized

### Advances in Proteomic Workflows for Systems Biology

Johan Malmstrom, Hookeun Lee, and Ruedi Aebersold. *Curr Opin
Biotechnol* **18(4):**378-384
(2007)

**Main Focus**

The article summarizes recent improvements as well as some principal
limitations of shortgun tandem mass spectrometry based proteomics.
Furthermore, it also briefly introduces steps of targeted driven
quantitative proteomics.

**Summary**

In recent years, great improvements have been made in all the parts of
non targeted mass spectrometry based proteomics including sample
preparation, data acquisition, data processing and analysis. In the
sample preparation process, with the introduction of IEF separation
method, resolution obtained from classical two dimensional
chromatography peptide separation is greatly improved. Improvements are
also made in the field of data quality which is increased by the
development of highly reproducible capillary chromatography methods and
quantitative analysis by stable isotope labeling method. High mass
resolution and accuracy could be achieved now by different types of mass
spectrometry such as TOF-TOF,Q-TOF in the data acquisition process.
Furthermore, different types of mass analyzers and ion sources have been
combined to increase the proteome coverage. With the development of
database search tools, the quality of proteomics data could be more
accurately assessed and estimated in the data processing and analysis
process.

Despite all these improvements achieved, limitations exist in shotgun
approaches. For example, shotgun MS datasets are extremely redundant
which greatly affect the identification of peptides present in proteomic
samples. The existence of semi-tryptic or non-tryptic peptides in
samples made the sample more complex. Saturation effect greatly reduces
the discovery rate of new proteins. Many peptides that detected by Mass
Spectrometry could not be identified, making it difficult to compare
sample to sample.

The limitations of shotgun approaches made the development of targeted
driven quantitative proteomics necessary. The first step of targeted
driven quantitative proteomics is protein and peptide selection. This
step could be finished both experimentally and computationally. For the
multiple reaction monitoring (MRM) and data analysis step, multiple
reaction monitoring was applied to proteomics data analysis. Relevance
to the course: this source is a brief overview of recent improvements in
targeted mass spectrometry (one method of proteomics) based proteomics
as well as some limitations. It also introduced another field of
proteomics: targeted driven quantitative proteomics.

**New Terms**

Electrospray ionization:A technique used in mass spectrometry to produce ions.It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized (http://en.wikipedia.org/wiki/Electrospray_ionization)\
Matrix-assisted laser desorption/ionization (MALDI):A soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as proteins,peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods(http://en.wikipedia.org/wiki/MALDI-TOF)\
PeptideAtlas:A multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments(http://www.peptideatlas.org/)\
Multiple reaction monitoring:MRM experiments, using a triple quadrupole instrument, are designed for obtaining the maximum sensitivity for detection of target compounds. This type of mass spectrometric experiment is widely used in detecting and quantifying drug and drug metabolites in the pharmaceutical industry(http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2291721)\
FT-ICR mass spectrometry:Fourier transform ion cyclotron resonance mass spectrometry, also known as Fourier transform mass spectrometry, is a type of mass analyzer (or mass spectrometer) for determining the mass-to-charge ratio (m/z) of ions based on the cyclotron frequency of the ions in a fixed magnetic field(http://en.wikipedia.org/wiki/Fourier_transform_ion_cyclotron_resonance)

**Course Relevance**

:   This source is about non targeted mass spectrometry and targeted
    approaches which are important methods in the identification of
    proteins(an important step in proteomics).

### Broad-Based Proteomic Strategies: A Practical Guide to Proteomics and Functional Screening

Graham David, Elliot Steven, Van Eyk Jennifer. \"Journal of
Physiology\" 563: 1-9
(2005)

**Main Focus**

This article summarizes what broad-based proteomics is and how one can
design a study using this global-view strategy. It first briefly looks
at the current technology in proteomics and then discusses how these
technologies can be incorporated into a study.

**Summary**

Proteomics as a field is becoming a very daunting one to enter because
many studies are getting lost in the complicated focused details. To
help assist with this challenge, a researcher can employ broad-based
proteomics. Broad-based proteomics is a strategy where careful planning
is employed upfront to answer a question about a proteome (for instance,
comparisons between a tissue in a diseased state and a normal state)
using the most appropriate and applicable technologies available. By
developing a strategy at the beginning of a proteomics study, possible
setbacks during the study are avoided.

The first step is to develop a general hypothesis that is specific to
the problem or issue that is being studied. Since proteomics mirrors
genomics, a proteomic study is increasingly difficult when the genome of
the model organism isn\'t known. For this reason, organisms where the
majority of the genome is known (80% or greater) should be chosen. Once
a proper organism has been chosen for study, the next factors to
consider are the type of data that will be generated and also the sample
source. Some proteomic methods yield qualitative data, while others
yield quantitative; so the type of data needed should be determined
before a method is chosen. At the same time, the source of the sample is
important in determining the extraction and purification methods.
Typical sample types include: urine, blood (plasma/serum) and mucosal
secretions. Protein concentration within the sample is important, and
one should expect reasonable extraction if the protein can be visualized
on a coomassie blue stained gel (\> 300 ng). The separation technique
chosen should reflect the characteristics of the protein(s) of choice
(hydrophobic vs hydrophilic, molecular mass, etc).

Another major factor in the planning process is estimating the
difficulty in the preparation of the fractioned sample for mass
spectrometry identification. Each mass spectrometry technique requires
varying degrees of preparation, and some are much more complicated than
others (2DE with MS/MS analysis requires greater preparation than HPLC
with MS, for instance). Since mass spectrometry is often the step where
a lot of proteomic studies encounter difficulty (both in preparation and
in interpretation of the results), it is very important to choose a
method that is appropriate for the protein sample.

With the advent of proteomic databases in recent years, bioinformatics
has had an increasing presence in proteomic studies. For this reason,
almost all proteomic studies should incorporate bioinformatics; and
consequently it\'s important for the research team to have some
bioinformatics knowledge. And depending on how much data will be
received at the end of the study (depending on the analysis methods
chosen), the research team can determine how much bioinformatic analysis
should be needed.

A final factor to consider is whether to bring in outside assistance or
to attempt the study in a more self-contained way. Keeping it
self-contained allows for the research team to keep its data integrated
and also keeps miscommunication to a minimum. Bringing in outside help,
on the other hand, could allow a researcher to tackle problems that
would be large and normally not solvable with a smaller team. While
bringing in outside assistance seems promising, it\'s important to not
lose control over the data and to make sure that the team is not spread
out trying to accomplish more than it can handle.

Since there are many ways to study a cell\'s proteome, careful planning
should be implemented at all stages of a proteomics study. Through
broad-based proteomics, a researcher can define a test plan before any
actual study is performed. And when used appropriately, this strategy
can lead to productive and efficient projects that will bring science
one step closer to understanding the proteome as a whole.

**New Terms**

Isoforms: A set of different proteins that form because of single nucleotide polymorphisms in the genomic sequence. ( <http://en.wikipedia.org/wiki/Isoform> )\
Single-nucleotide polymorphism (SNP): a DNA sequence variation occurring when a single nucleotide in the genome differs between members of a species. ( <http://en.wikipedia.org/wiki/Single_nucleotide_polymorphism> )\
Post-translational modification (PTM): the chemical modification of a protein after it has been translated. It is usualy one of the last steps in protein biosynthesis for most proteins. ( <http://en.wikipedia.org/wiki/Posttranslational_modification> )\
Subproteome: a subfractioned subset of the proteome. Often these are linked to area of the cell (organelle for instance) or by chemical properties.\
Peptide mass fingerprinting (PMF): an analytical technique for protein identification. The unknown protein of interest is first cleaved into smaller peptides and after mass is determined using mass spectrometry, their masses are compared to either a database containing known protein sequences or a genome. ( <http://en.wikipedia.org/wiki/Peptide_mass_fingerprinting> )

**Course Relevance**

:   This article is relevant because a global view of proteomics is
    becoming more important. As the wealth of information about proteins
    expands, understanding the proteome from a broad viewpoint is
    becoming more and more useful.

## Websites Summarized

### The Association of Bimolecular Resource Facilities: Proteomics Research Group (PRG)

Website committee: Pamela Scott Adams, Michelle Detwiler, David Mohr
James Ee, Dr. Xiaolog Yang, Dr. Len Packman, Dr. Anthony Yeung,
<http://www.abrf.org/index.cfm/group.show/Proteomics.34.htm#R_3>
(3/25/09)

**Main Focus**

This web page is about how the Association of Bimolecular Resource
Facilities relates to proteomics. Of particular importance is the
Proteomics Research Group within the ABRF.

**Summary**

The Association of Bimolecular Resource Facilities (ABRF) is an
international association of research facilities and laboratories that
is focused on core research in Biotechnology. The association encourages
the sharing of information through conferences, a quarterly journal, and
group studies. The ABRF has a heavy influence on the field of
proteomics, and there are five main research groups (RG) that deal with
proteomics in some way: Protein Expression (PERG), Protein Sequencing
(PSRG), Protein Informatics (iPRG), Proteomics (PRG), and Proteomics
Standards (sPRG).

Of particular importance, the Proteomics Research Group allows for
researchers throughout the world in the field of proteomics to share
their protein analysis information freely. Obviously, since
understanding the proteome is about bringing together information on
many different proteins (which is information that requires a great
amount of effort/time/money to achieve), the sharing of
protein/subproteomic information is imperative to beginning to
understand a proteome in its entirety. This website has numerous links
to studies performed by research groups throughout the world.

**New Terms**

De Novo Peptide Sequencing: Peptide sequencing that is performed without any prior knowledge of the amino acid sequence. (http://www.ionsource.com/tutorial/DeNovo/DeNovoTOC.htm)\
Quantitative Proteomics: Has the goal of obtaining quantitative information about all the proteins in a particular sample. This is useful because it allows for one to see the differences in protein samples. (http://en.wikipedia.org/wiki/Quantitative_proteomics)

**Course Relevance**

:   This is an overview of the Association of Biomolecular Resource
    Facilities (ABRG) and how it relates to proteomics. There is a great
    deal of relevant information on this website that those in
    proteomics will find useful.

### Introduction to Proteomics

Writer/Producer: Rick Groleau,Subject Matter Expert: Hanno Steen,
PhD,Designer: Peggy Recinos,Developer: Jeffrey Testa,
<http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.html>
(28 March 2009)

![](Flowchart_of_proteomics.gif "Flowchart_of_proteomics.gif"){width="250"
height="350"}

**Main Focus**

This web page is about the importance and challenges in proteomics. It
also introduces major steps of proteomics briefly.

**Summary**

Proteomics is important for us to understand biological processes since
all the functions are accomplished by proteins in cell.But as the number
of proteins are so large and amino acids(which are units of protein) are
so small, the study is quite challenging.There are five steps to analyze
protein sequences: sample preparation,separation,ionization,mass
spectrometry and informatics.First of all, we obtain cells and extract
proteins from the cells.Then we use methods such as 2D electrophoresis
to separate proteins. Next, we use protease to cut proteins into
peptides.Mass spectrometry allows us to identify individual peptides as
well as peptides fragments.Finally, by interpreting the data, we are
able to determine the sequence of proteins.

**New Terms**

Biopsy: A biopsy is a medical test involving the removal of cells or tissues for examination. It is the removal of tissue from a living subject to determine the presence or extent of a disease(http://en.wikipedia.org/wiki/Biopsy)\
TOF: The time of flight (TOF) describes the method used to measure the time that it takes for a particle, object or stream to reach a detector while traveling over a known distance(http://en.wikipedia.org/wiki/Time-of-flight)\
Quadrupole mass spectrometry: The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry.It consists of 4 circular rods, set perfectly parallel to each other.In a quadrupole mass spectrometer the quadrupole mass analyzer is the component of the instrument responsible for filtering sample ions, based on their mass-to-charge ratio (m/z).Ions are separated in a quadrupole based on the stability of their trajectories in the oscillating electric fields that are applied to the rods(http://en.wikipedia.org/wiki/Quadrupole_mass_analyzer)\
Electronspray ionization: Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions.It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized(http://en.wikipedia.org/wiki/Electrospray_ionization)\
Dalton: Dalton is the unit of measurement for atomic mass. One Dalton is equal to 1/12th the mass of one atom of carbon12(http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P1.html)

**Course Relevance**

:   This is an overview of proteomics. It summarizes the procedures and
    importance of proteomics very briefly.

### Introduction to Proteomics

Institute of Biology and Medical Genetics of the First Faculty of
Medicine of Charles University and the General Teaching
Hospital,http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm (6 April
2009)

![](proteomics.jpg "proteomics.jpg"){width="250" height="350"}

**Main Focus**

This website discusses the aims and definitions of proteomics. It also
introduces two important methods in proteomcis studies - 2D protein
electrophoresis and mass spectrometry as well as proteomics in medicine

**Summary**

Proteomics is a broad field which includes expression proteomics,
protein distribution in subcellular compartments of the
organelles,post-translational modifications of the proteins,structural
proteomics and functional proteomics, clinical proteomics and so on.
Even though analysis of the expression on transcripts level is possible
with the introduction of RNA/cDNA microarray, proteomics is still
important since not all mRNA will be translated and the processes such
as RNA splicing, posttranslational protein modifications exist.

Two-dimensional (2D) protein electrophoresis is commonly used to
separate proteins based on their PI and mass. Mass spectrometry is an
important method in proteomics since it cannot only be used for protein
identification but can also be used for protein posttranslational
modification analysis.

One of the major application of proteomics in medicine is the
identification of markers in all the steps to treat diseases. Other
applications include drug discovery and pharmacoproteomics.

**New Terms**

Human Proteome Organization(HUPO): The Human Proteome Organisation (HUPO) is an international scientific organization representing and promoting proteomics through international cooperation and collaborations by fostering the development of new technologies, techniques and training(http://www.hupo.org/)\
structural proteomics: Structural proteomics is an international collaboration project for solving 3D protein structures at a proteome scale(http://en.wikipedia.org/wiki/Structural_proteomics)\
Swedish Human Protein Atlas: The Swedish Human Protein Atlas program (HPA), funded by the (non-profit) Knut and Alice Wallenberg Foundation, invites submission of antibodies from both academic and commercial sources to be included in the human protein atlas (http://www.proteinatlas.org)\
Posttranslational modification (PTM): Posttranslational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis for many proteins(http://en.wikipedia.org/wiki/Posttranslational_modification)\
Isoelectric point: Isoelectric point is such a pH value, where the overall protein charge equals to zero(http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm)

**Course Relevance**

:   This website gives brief definition and aims of proteomics.It also
    introduces principles of 2D-electrophoresis and mass spectrometry
    which are important methods in proteomics.

\
Contact: jxr0084@rit.edu, sfl9376@rit.edu

## References

-   Johan Malmstrom, Hookeun Lee, and Ruedi Aebersold. \"Advances in
    Proteomic Workflows for Systems
    Biology\"*Curr
    Opin Biotechnol* **18(4):**378-384 (2007)\
-   Rick Groleau,Hanno Steen,Peggy Recinos,Jeffrey Testa. \"Introduction
    to Proteomics\"
    <http://www.childrenshospital.org/cfapps/research/data_admin/Site602/mainpageS602P0.html>
    (28 March 2009)
-   Institute of Biology and Medical Genetics of the First Faculty of
    Medicine of Charles University and the General Teaching Hospital.
    \"Introduction to Proteomics\"
    <http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm> (06 April 2009)


# Proteomics/Protein Sample Preparation

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## Introduction

As technological advances are made in the field of proteomics, it is
seen that advances are necessary in the preparation of protein samples
prior to any particular procedure. A number of issues arise in this
respect; including sample clean-up, fractionation, enrichment, and the
also sample condition optimization. Considerations of this nature can be
crucial in obtaining relevant results from an experiment; some so much
so that experts feel the field of proteomics is currently being limited
by the lack of significant advancement in sample preparation techniques.

This facet of proteomics is becoming particularly critical in the case
of high throughput protocols where the necessary conditions of a sample
in one stage may directly conflict with the efficacy of a second stage.
For example, during the initial step in 2D electrophoresis, isoelectric
focusing, all proteins in a sample are given a net charge of zero; while
the second step, gel electrophoresis, requires a negative charge on all
products in the sample in order to induce movement through the gel
matrix.

Many companies offer pre-packaged kits that will allow you to prepare
samples for many different techniques. They also offer many protein
samples, and other protein technologies. Many of these companies are
also on the forefront of protein analysis technology. Some examples are:

1.  www.millipore.com
2.  www.bio-rad.com
3.  www.qiagen.com
4.  www.gelifesciences.com
5.  www.invitrogen.com
6.  www.eksigent.com
7.  www.agilent.com
8.  www.beckmancoulter.com
9.  www.caliperls.com
10. www.glygen.com
11. www.piercenet.com
12. www.bioproximity.com

This section is part of an ongoing project at the Rochester Institute
of Technology, involving the
Bioinformatics department. Currently
the project is being worked on by a Proteomics Class taught by Dr. Paul
Craig.

## Bibliography

-   Jörg von Hagen, VCH-Wiley 2008 Proteomics Sample Preparation.


# Proteomics/Plant Proteomics about Two Dimensional Gel Electrophoresis

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This page contains protocols that are frequently used in proteomics. You
are welcome to add protocols this chapter.

1.  /Plant Proteomics about Two Dimensional Gel
    Electrophoresis/


# Proteomics/Protein Separations - Chromatography

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Chapter written by: Laura Grell and Alexander Butarbutar\
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions\
Chapter modified by Kai Burnett and Dalia Ghoneim\
Contact kab9783@rit.edu or dxg6098@rit.edu \_\_FORCETOC\_\_

## Introduction

!HPLC{width="350"}

(Res1)
To obtain a pure protein sample, a protein must be isolated from all
other proteins and cellular components. This can prove to be a difficult
task as a single protein often makes up only 1% of the total protein
concentration of a cell. Therefore 99% of the protein components of a
sample must be removed before it can be classified as pure. A task that
is equally challenging is keeping the protein in its active form. When
we purify proteins we remove them from their natural environments. As a
result, it is necessary to simulate the pH, salt concentration and
reducing conditions in which they are normally found. In the process of
obtaining an active and pure sample we want to minimize the number of
steps taken in order to maximize the yield at the end of the separation.
Finally, since proteins are made with the intention of only functioning
for a short period of time, it is also critical to obtain our sample as
quickly as possible. All these components of protein separations can be
successfully achieved by a group of separation methods collectively
known as
chromatography.

There are several properties of proteins that can be taken advantage of
to separate proteins. Different types of chromatography take advantage
of different properties. Proteins can be separated by:

-   size
-   shape
-   hydrophobicity
-   affinity to molecules
-   charge

!A Typical Column{width="350"}    In
this chapter several different chromatographic methods will be
introduced and described. While the methods outlined below all use
different characteristics of proteins to separate proteins from one
another, they all utilize an insoluble stationary phase and a mobile
phase that passes over it. The mobile phase is commonly a liquid
solution. It contains the protein we want to isolate. The stationary
phase on the other hand is made up of a grouping of beads, usually based
on a carbohydrate or acrylamide derivative, that are bound to ionically
charged species, hydrophobic characters, or affinity ligands. Much of
the success of chromatography is associated with the selection of an
appropriate stationary phase.

   In column
chromatography,
when a protein sample is applied to the column, it equilibrates between
the stationary phase and the mobile phase. Depending on the type of
chromatography, proteins with certain characteristics will bind to the
stationary phase while those lacking the sought characteristics will
remain in the mobile phase and pass through the column. For example in
ion exchange chromatography, a positively charged protein would bind to
a negatively charged stationary phase, while the negatively charge
protein will be eluted from the column with the mobile phase. The final
step involves displacing the protein from the stationary phase, also
known as elution, by introducing a particle which will compete with the
protein binding site on the stationary phase. Today various commercial
column are readily available, specifically
Bio-Rad,
Sigma-Aldrich,GE
Healthcare
and Omnifit offers a
wide variety of chromatography column.

!Common Chromatogram RIT Ion Exchange
Site

The image above is a chromatogram that shows the results of a separation
based on signals interpreted by a detector.

t~m~ - the time required for the mobile phase to travel the entire
length of the column

t~r~ - the time required for a specific protein to elute from the column

## Resources

1.  Craig, P. Designing a
    Separation
2.  Florida State University, \"Chromatography\" Michael Blaber\'s
    Biochemistry
    Lab
3.  GE Healthcare - 1 -
    \[<http://www.gelifesciences.com/protein-purification>?\]
4.  BioPharm International Guide Basics of
    Chromatography
    (2003).
5.  Bio-Rad Chromatography Protein
    Purification
    \|\| Green Fluorescent Protein Chromatography
    Kit
6.  BioForum Topics In
    Chromatography
7.  Journal of Chromatographic Science
8.  M.Isabel Pedraza Mayer Chromatography
    Database

## References

1.  Harris, D.C. \"Quantitative Chemical Analysis; 6th Edition\", W.H.
    Freeman and Company: New York..
2.  Patrick McKay An Introduction to
    Chromatography
    Senior Research Associate, Department of Recovery Sciences,
    Genentech, Inc \*.

.\* Denotes Free Article


# Proteomics/Protein Separations- Electrophoresis

CurrentPage=Introduction to Electrophoresis|
PrevPage=[[Proteomics/Protein Separations - Chromatography|Previous Chapter - Protein Separations: Chromatography]]|
NextPage=[[Proteomics/Protein Separations- Electrophoresis/Gel Electrophoresis|Gel Electrophoresis]]}}
```
\_\_TOC\_\_

## Definitions

e•lec•tro•pho•re•sis (ĭ-lĕk\'trō-fə-rē\'sĭs) n.
\
1) The migration of charged colloidal particles or molecules through a
solution under the influence of an applied electric field usually
provided by immersed electrodes. Also called cataphoresis.\
2) A method of separating substances, especially proteins, and analyzing
molecular structure based on the rate of movement of each component in a
colloidal suspension while under the influence of an electric field.

\
an•a•lyte (a-nə-līt) n. \
A chemical substance that is the subject of chemical analysis.

!Separation under
electrophoresis

## Electrophoresis Theory

Separation by electrophoresis depends on differences in the migration
velocity of ions or solutes through a given medium in an electric field.
The electrophoretic migration velocity of an analyte is:

`<big>`{=html} $v_p = \mu_p E$ `</big>`{=html}

where E is the electric field strength and $\mu_p$ is the
electrophoretic mobility.

\
The electrophoretic mobility is inversely proportional to frictional
forces in the buffer, and directly proportional to the ionic charge of
the analyte. The forces of friction against an analyte are dependent on
the analyte\'s size and the viscosity (η) of the medium. Analytes with
different frictional forces or different charges will separate from one
another when they move through a buffer. At a given pH, the
electrophoretic mobility of an analyte is:

$\mu_p = \frac{z}{6\pi \eta r}$

where r is the radius of the analyte and z is the net charge of the
analyte.

\
Differences in the charge to size ratio of analytes causes differences
in electrophoretic mobility. Small, highly charged analytes have greater
mobility, whereas large, less charged analytes have lower mobility.
Anionic surfactants are often used to denature proteins before
electrophoresis. This gives all proteins approximately the same charge.
Since their charges are all equal their mobility is now a function of
only their masses. Electrophoretic mobility is an indication of an
analyte\'s migration velocity in a given medium. The net force acting on
an analyte is the balance of two forces: the electrical force acting in
favor of motion, and the frictional force acting against motion. These
two forces remain steady during electrophoresis. Therefore,
electrophoretic mobility is a constant for a given analyte under a given
set of conditions.

## Applications of Electrophoresis

Electrophoresis has a wide variety of applications in proteomics,
forensics, molecular biology, genetics, biochemistry, and microbiology.

One of the most common uses of electrophoresis is to analyze
differential expression of genes. Healthy and diseased cells can be
identified by differences in the electrophoretic patterns of their
proteins. Proteins themselves can also be characterized in this way, and
some sense of their structure can be derived from the masses of
fragments inside the gel.

There are many different types of electrophoresis, and each can be used
for something different. Two-dimensional (2-D) electrophoresis, for
example, has the ability to discern many more proteins than most of its
contemporaries. Many of these methods will be discussed in detail
throughout this chapter.

## References

1.      The American Heritage Dictionary of the English Language, Fourth
    Edition. <http://www.bartleby.com/61/>

2.      The Merriam-Webster Online Dictionary. <http://www.m-w.com>

3.      Mans, Andreas et al. _Bioanalytical
    Chemistry._ Imperial College Press, 2004.

4.      Twyman, Richard. \"Two-dimensional polyacrylamide gel
    electrophoresis.\" <http://genome.wellcome.ac.uk/doc_wtd021045.html>

------------------------------------------------------------------------

Next page
\>


# Proteomics/Protein Separations - Centrifugation

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## Introduction to Centrifugation

!Tabletop
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Centrifugation is one of the
most important and widely applied research techniques in
biochemistry, cellular and molecular
biology, and in medicine. In the field of
proteomics it plays a vital role in the fundamental and necessary
process of isolating proteins. This process begins with intact cells or
tissues. Before the proteins can be obtained, the cells must be broken
open by processes such as snap freezing, sonication, homogenization by
high pressure, or grinding with liquid nitrogen. Once the cells have
been opened up all of their contents; including cell membranes, RNA,
DNA, and organelles will be mixed in the solvent with the proteins.
Centrifugation is probably the most commonly used method for separating
out all the non protein material. Within the centrifuge samples are spun
at high speeds and the resulting force causes particles to separate
based on their density.

## Uses of Centrifugation

Centrifugation is capable of:

-   Removing cells or other suspended particles from their surrounding
    milieu on either a batch or a continous-flow basis

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-   Separating from dispersed tissue the various subcellular organelles
    including nuclei, mitochondria, cholorplasts, golgi bodies,
    lysosomes, peroxisomes, glyoxysomes, plasma membranes, endoplasmic
    reticulum, polysomes, and ribosomal subunits.

Once the mixture of proteins has been isolated using centrifugation the
scientist is then able to use one of several methods to separate out
individual proteins for further study. For more information on protein
purification/separation see Protein Separations --
Chromatography
and Protein Separations--
Electrophoresis.

Next section: /History of the
Centrifuge/

## References

### Subscription Based References

1.  Sheeler, P. \"Centrifugation in Biology and Medical Science.\" Dept
    of Biology, California State University, Northridge, California

### Open Access References

1.  Claude, A. & Potter, J. S. \"Isolation of Chromatin Threads From
    The Reasting Nucleus of Leukemic
    Cells\" The Journal of
    Experimental Medicine.


# Proteomics/Emerging and Miscellaneous Proteomics Technologies

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\_\_FORCETOC\_\_

# Emerging and Miscellaneous Technologies in Proteomics

This part of the book will be an area where proteomics techniques that
have been newly developed will be discussed. Techniques that do not
currently fit into any other part of the book can also be added to this
page. As chapters or sections are added elsewhere that discuss these
techniques in the context of a greater proteomics problem, the
information on this page will be moved to those pages.

## X-Ray Tomography

**Description and Discussion of X-ray Tomography.**

A new branch of X-ray microscopy is being used in proteomics analysis.
This is called X-ray Tomography. This method uses projected images to
calculate and reconstruct a 3D object. This technology is being used in
proteomics to determine the location of labeled proteins or large
complexes within a cell. This technique can also be used in conjunction
with images of cells from light based microscopes to help identify where
a protein is located and how this location factors in to its function
and identification.

## Introduction to Proteoinformatics

Proteoinformatics is the use of bioinformatics and computational biology
techniques solely within the realm of protein identification and
proteomics. Proteoinformatics is currently in its infancy and the
largest work being done is on standardizing databases and data
submission. Other proteoinformatic work is being done on the image
analysis of 2D gels and other images in proteomics used to help identify
and annotate proteins in the proteome.

### Protein Identification Database

#### What are Protein Identification Databases?

Protein Identifications Databases such as ProFound at Rockefeller
University and Protein Prospector at the UCSF Mass Spectrometry Facility
are used to help identify proteins found with proteomics techniques such
as mass spectrometry. Digestion of proteins into peptide fragments
allows each protein to break apart in a different way, resulting in a
unique peptide fingerprint that can be used to identify the protein. The
masses of these fragments as well as the molecular weights and
isoelectric points are what is stored in many of these databases. This
data can be used to perform high-throughput protein identification.

#### The Future of Protein Identification Databases

In order to continue advancing the cause of mapping the human proteome,
international databases need to be established which integrate both
transcriptome and proteome data. The Human Proteome Organization is
currently working on establishing a defined standard for data submission
and annotation for the many different proteomics techniques currently
used to identify and annotate proteins.

### New Techniques in Image Analysis

According to the Image Analysis Wikipedia page, \"Image analysis is the
extraction of meaningful information from images.\" In terms of
Proteomics, image analysis can be used to compare different images
generated using proteomics techniques, such as 2D-PAGE gel images. New
programs are being developed that will help to optomize and automate the
process of locating a protein spot between two gel images in order to
identify the differences between 2D-PAGE gels. Other programs can be
used to help clean up and remove variability between these images as
well.

## Laser Capture Microdissection

!Diagram of Laser Capture
Microdissection
Laser capture microdissection or LCM is a process that isolates and
removes distinct populations from a tissue. This will facilitate the
comparison of diseased tissue with normal tissue from an organism.

In LCM, an infrared laser beam melts a thermosensitive polymer film that
traps a specific group of cells. This polymer film is then extracted and
moved to a test tube where an extraction buffer is used to remove the
groups of cells for more advanced proteomics analysis such as 2D-PAGE,
Ion Chromatography, etc. This technology will become more useful as
systems with higher sensitivity for analysis of smaller amounts of
tissue become developed and realized.

## Proteomic Complex Detection using Sedimentation

Approaches such as TAP tagging, which require the addition of fusion
proteins, can interfere with protein interactions that would have
normally occurred. Many times it takes a great deal of work to express
these tagged proteins, so this technique is used to give evidence that
there is a stable protein complex detected early on in the proteomics
experiment before more laborious approaches are used to isolate and
identify the protein complexes of interest. Issues also occur in MS and
2D Gel processes where one cannot be sure that a portion of a gel spot
is the desired protein because multiple proteins could be traveling
together in that spot as a complex.

This is where proteomic complex detection using sedimentation (ProCoDeS)
is applicable. ProCoDeS is a technique for the high-throughput
identification of both soluble and membrane proteins that are found in
stable complexes. Relative sizes of protein complexes are estimated via
their sedimentation in a gradient. In this case a rate zonal gradient or
RZG is used to better estimate the relative size of protein complexes.
The distribution of a protein of interest in this sedimentation can be
detected using classic techniques such as Western Blotting or newer
techniques such as ICAT. This can be done for a large number of
proteins. Thus, ProCoDeS can be used to identify stable protein
complexes. ProCoDeS is especially well suited for the screening of
unrefined cellular material to help find new proteins that cannot be
discovered because they exist in protein complexes such as proteins
found in protein membranes.

Next Chapter: Protein Identification - Mass
Spectrometry

## References

1.  Hartman, N. T., et al. \"Proteomic Complex Detection Using
    Sedimentation\"
    Anal. Chem., 79, 5, 2078 - 2083, 2007.
2.  NCT Proteomics Group \"Emerging
    Technologies\"
    National Institutes of Environmental Health Sciences
3.  NCT Proteomics Group
    \"ProteoInformatics\"
    National Institutes of Environmental Health Sciences
4.  \"Wikipedia: Image
    Analysis
5.  \"Wikipedia: Proteomics
6.  \"Wikipedia: X-ray
    Tomography


# Proteomics/Protein Identification - Mass Spectrometry

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!MALDI TOF MS

## Introduction

-   **Mass Spectrometry Overview**

Mass spectrometry is a technique in which gas phase molecules are
ionized and their mass-to-charge ratio is measured by observing
acceleration differences of ions when an electric field is applied.
Lighter ions will accelerate faster and be detected first. If the mass
is measured with precision then the composition of the molecule can be
identified. In the case of proteins, the sequence can be identified.
Most samples submitted to mass Spectrometry are a mixture of compounds.
A spectrum is acquired to give the mass-to-charge ratio of all compounds
in the sample. Mass spectrometry is also known as \'mass spec\' or MS
for short. Mass spectrometry throws light on molecular mechanisms within
cellular systems. It is used for identifying proteins, functional
interactions, and it further allows for determination of subunits. Other
molecules in cells such as lipid components can also be defined.

A mass spectrometer is composed of several different parts: a source
that ionizes the sample, the analyzer that separates the ions based on
mass-to-charge ratio, a detector that \"sees\" the ions, and a data
system to process and analyze the results. You can also measure relative
abundance of an ion using mass spectrometry. Different compounds have
differential ionization capabilities and therefore intensity of your ion
is not a direct correlation to concentration.

Mass spectrometry can be a high throughput analytical method due to the
ability for a mass spectrum to be measured rapidly and with minimal
sample handling as compared to gel methods.

It is an analytical method which has a variety of uses outside of
proteomics, such as
isotope and dating, trace gas analysis, atomic location mapping,
pollutant detection, and space exploration

-   **History of Mass Spectrometry**

The history of this technique finds its roots in the first studies of
gas excitation in a charged environment, more than 100 years ago. This
pioneering work led to the identification of two isotopes of neon
(neon-20 and neon-22) via mass to charge ration discrimination by J.J
Thomson in 1913. Over the next fifty years
the fundamental basis of the technique was further developed. After the
coupling of gas chromatography to Mass Spectroscopy in 1959 by
researches at Dow Chemical, the full potential of the technique as a
highly accurate, quantitative method for exploring compounds was
realized, spurring a wave of developments which continue to the present
day. The precision of mass spectrometry led to the discovery of
isotopes.

\
\***Implications of Mass Spectrometry for Proteomics Applications** The
technique of mass spectrometry is a valuable tool in the field of
proteomics. It can be used to identify proteins through variations of
mass spectrometry techniques. The most common first approach to
proteomics is a bottom-up approach in which the protein is digested by a
protease, such a trypsin, and the peptides are then analyzed by peptide
mass fingerprinting, collision-induced
dissociation, tandem MS,
and electron capture
dissociation. Once the
peptides masses have been determined the mass list can be sent to a
database, such as MASCOT, where the list is compared to the masses of
all known peptides. If enough peptides match that of a known protein you
can identify your protein. If the masses of your peptides do not match a
known protein you can sequence your peptide by de novo sequencing using
MS/MS methods; where you isolate your peptide and break it along the
peptide bond backbone forming y and b ions from which you can determine
the sequence. The advantages of the bottom-up approach are that the
small size of tryptic peptide ions is easy to handle biochemically than
entire protein ions because of their relatively small masses that are
easier to be determine. Beside bottom-up approach, another approach is
top-down. In top-down approach, the complete proteins are directly
analyzed by using mass spectrometer without solution digestion as
bottom-up does. The advantages of the top-down approach are that it can
sometime provide the complete coverage of the protein. But since whole
proteins are hard to handle biochemically compared to small peptide
pieces, it makes top-down approach difficult to analyze.

Another use of mass spectrometry in proteomics is protein
quantification. By labeling proteins with stable heavier isotopes you
can in turn determine the relative abundance of proteins. Companies now
produce kits, such as iTRAQ (Applied
Biosystems),
in order to do this at a high-throughput level.

One of the most powerful ways to identify a biological molecule is to
determine its molecular mass together with the masses of its component
building blocks after fragmentation. There are two dominant methods for
doing this. The first is electrospray ionization (ESI), in which the
ions of interest are formed from solution by applying a high electric
field. This is done by applying a high electric field to the tip of a
capillary, from which the solution will pass through. The sample will be
sprayed into the electric field along with a flow of nitrogen to promote
desolvation. Droplets will form and will evaporate in a vacuumed area.
This causes an increase in charge on the droplets and the ions are now
said to be multiply charged. These multiply charged ions can now enter
the analyzer. ESI is a method of choice because of the following
properties: (1)The \"softness\" of the phase conversion process allows
very fragile molecules to be ionized intact and even in some
non-covalent interactions to be preserved for MS analysis. (2)The
eluting fractions through liquid chromatography can then be sprayed into
the mass spectrometer, allowing for the further analysis of mixtures.
(3)The production of multiply charged ions allow for the measurement of
high-mass biopolymers. Multiple charges on the molecule will reduce its
mass to charge ratio when compared to a single charged molecule.
Multiple charges on a molecule also allows for improved fragmentation
which in turn allows for a better determination of structure. The second
is matrix-assisted laser desorption/ionization (MALDI) in which the
molecular ions of interest are formed by pulses of laser light impacting
on the sample isolated within an excess of matrix molecules. This
enables the determination of masses of large biomolecules and synthetic
polymers greater than 200,000 Daltons without degradation of the
molecule of interest. The advantages of MALDI are its robustness, high
speed, and relative immunity to contaminants and biochemical buffers.

A type of mass spectrometer often used with MALD is TOF or Time of
Flight mass spectrometry. This enables fast and accurate molar mass
determination along with sequencing repeated units and recognizing
polymer additives and impurities. This technique is based on an
ultraviolet absorbing matrix where the matrix and polymer are mixed
together along with excess matrix and a solvent to prevent aggregation
of the polymer. This mixture is then placed on the tip of a probe; then
the solvent is removed while under vacuum conditions. This creates
co-crystallized polymer molecules that are dispersed homogeneously
within the matrix. A pulsing laser beam is set to an appropriate
frequency and energy is shot to the matrix, which becomes partially
vaporized. In turn the homogeneously dispersed polymer within the matrix
is carried into the vapor phase and becomes charged. To obtain a superb
signal-to-noise ratio, multiple laser shots are executed. The shapes of
the peaks are improved and the molar masses determined are more
accurate. Fianlly, in the TOF analyzer the molecules from a sample are
imparted identical translational kinetic energies because of the
electrical potential energy difference. These ionic molecules travel
down an evacuated tube with no electrical field and of the same
distance. The smallest ions arrive first at the detector, which produces
a signal for each ion. The cumulative data from multiple laser shots
yield a TOF mass spectrum, which translates the detector signal into a
function of time, which in turn can be used to calculate the mass of the
ion.

In addition to these ionization techniques, highly powerful mass
analyzers have been developed. These analyzers measure the mass/charge
ratio of intact ionized biomolecules, as well as their fragmentation
spectra, with high accuracy and high speed. The measurement of
fragmentation spectra is called tandem MS or MS/MS. In conjunction with
single stage MS (with intact precursor ions) tandem MS can be utilized
to help elucidate a protein since the problem of elucidation will reduce
to assembling the puzzle pieces of the fragmented protein.

## References

1.  American Society for Mass Spectrometry - What is MS?,
    <http://www.asms.org/whatisms/p4.html>
2.  Mass Spectrometry in the Postgenomic Era
3.  Annual Review of Biochemistry Vol. 80: 239-246 (Volume publication
    date July 2011) DOI: 10.1146/annurev-biochem-110810-095744
    <https://ted.ucsd.edu/webapps/portal/frameset.jsp?tab_tab_group_id=_2_1&url=%2Fwebapps%2Fblackboard%2Fexecute%2Flauncher%3Ftype%3DCourse%26id%3D_767_1%26url%3D>
4.  University of Illinois at Urbana-Champaign School of Chemical
    Sciences <http://scs.illinois.edu/massSpec/ion/esi.php>
5.  University of Southern Mississippi School of Polymers and High
    Performance Materials <http://www.psrc.usm.edu/mauritz/maldi.html>

fr:Chimie organique/Spectrométrie de
masse


# Proteomics/Protein Primary Structure

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## Introduction


# Proteomics/Post-translational Modification

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## Introduction

I. Definition

`    A. Spontaneous or enzymatic alteration to one or more of a protein's amino acids`\
`    B. Most often manifests as an addition or deletion to a side-chain`\
`    C. Can occur at any point during or following full translation of a protein`\
`    D. Often drastically effects overall structure and function of protein and associated complexes`\
`    E. Are highly conserved among all living organisms`

II\. Types of modifications

`    A. Acetylation`\
`    B. Amidation/Deamidation`\
`    C. Glycosylation`\
`    D. Oxidation`\
`         i. S-Glutathionylation`\
`         ii. S-Nitrosylation`\
`    E. Phosphorylation`\
`         i. Histidine`\
`         ii. Serine`\
`         iii. Theronine`\
`         iv. Tyrosine`\
`    F. Proteolysis`\
`    G. Ubiquitinylation/SUMOylation`\
`    H. Others?`

III\. Manipulating in-vivo modifications

`    A. Modifications can be prevented or induced in organism, tissue, and cell based model systems`\
`    B. May allow for the detection of target proteins or dissection of related processes and pathways`\
`    C. Exogenous introduction of stimuli`\
`         i. endocrine/paracrine signals (aka hormones)`\
`         ii. environmental (temp, UV, heavy-metals, peroxide, etc.)`\
`         iii. antigens (virus, bacteria, allergens, lysates, etc.)`\
`         iv. chemical/medicinal activators and inhibitors`\
`    D. Genetic Approaches`\
`         i. deletion, mutation, or reorganization of genetic elements (enhancers, promoters, genes, etc.)`\
`         ii. gene inactivation or silencing by nucleic acid hybridization`\
`         iii. transgenics (tansformations and transfections)`\
`    E. Advantages and Draw-backs`\
`         i. Qualitative but often hard to quantify`\
`         ii. Often expolaratory in nature (observe and report)`\
`         iii. Often very large scale in terms of available data`

IV\. In vitro reconstitution strategies

`    A. Often provide a more quantitative, in depth analysis of a particular post-translational modification`\
`    B. The protein in question is added to a reaction with the appropriate reagents and/or enzymes`\
`    C. Reactions can be followed in real-time more readily`

V. Methods of detection

`    A. Most commonly used detection method of known modifications is through immuno/Western blotting`\
`    B. Unexpected or novel modifications can be detected with a variety of analytical techniques, most notably mass spectrometry`\
`    C. The best specific method of detection depends on many factors including the stability, frequency, and scale of the modification(s)`\
`    D. In practice ease and cost dictate which methods are used first while the more exhaustive, cumbersome, or expensive methods follow as needed`


# Proteomics/Protein - Protein Interactions

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Chapter edited and updated by: Poulami Barman and Sarah Allen

Contact pxb2979@rit.edu, sea3016@rit.edu

## Introduction

Protein interaction is crucial for every organism. Most proteins
function through interaction with other molecules, and often with other
proteins. Enzymes interact with their substrates, inhibitors interact
with enzymes, transport proteins interact with structural proteins,
hormones interact with receptors -- and that's just a few of the
interactions that happen in a cell. Some proteins are composed of more
than one polypeptide chain, and the interactions between the different
peptides are necessary for the whole protein to function. Since they are
so essential, protein-protein interactions are an important topic for
scientists to understand.

There are many characteristics of a protein-protein interaction that are
important. Obviously, it is important to know **which proteins are
interacting**. In many experiments and computational studies, the focus
is on interactions between two different proteins. However, you can have
one protein interacting with other copies of itself (oligomerization),
or three or more different proteins interacting. The
**stoichiometry** of the
interaction is also important -- that is, how many of each protein
involved are present in a given reaction. Some protein interactions are
stronger than others, because they bind together more tightly. The
strength of binding is known as **affinity**. Proteins will only bind
each other spontaneously if it is energetically favorable. **Energy**
changes during binding are another important aspect of protein
interactions. Many of the computational tools that predict interactions
are based on the energy of interactions.

In recent years there has been a strong focus on predicting protein
interactions computationally. Predicting the interactions can help
scientists predict pathways in the cell, potential drugs and
antibiotics, and protein functions. However, it has been an ongoing
challenge to decipher those interactions. Proteins are large molecules,
and binding between them often involves many atoms and a variety of
interaction types, including hydrogen
bonds, hydrophobic
interactions,
salt bridges,
and more. Proteins are also dynamic, with many of their bonds able to
stretch and rotate leading to different conformations. Therefore,
predicting protein-protein interactions requires a good knowledge of the
chemistry and physics involved in the interactions.

This chapter discusses the characteristics of protein-protein
interactions, how they are determined experimentally, and how they are
predicted computationally. It also contains a list of databases where
you can explore known and predicted protein interactions. The links
above will lead you to the various sections.


# Proteomics/Protein Chips

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**Introduction:** \_\_TOC\_\_

## Introduction

!A DNA microarray as seen through a microscope. Protein chips look
identical, except each spot corresponds to one of the organism\'s
thousands of proteins, instead of one of it\'s genes. The intensity of
the dot indicates the amount of protein
present.

Protein chips, also referred to
as protein arrays or protein microarrays, are modeled after DNA
microarrays. The success of DNA
microarrays in large-scale genomic experiments inspired researchers to
develop similar technology to enable large-scale, high-throughput
proteomic experiments. Protein chips enable researchers to quickly and
easily survey the entire proteome of a cell within an organism. They
also allow researchers to automate and parallelize protein experiments.

Protein chips were first developed in 2000 by researchers at Harvard
University.[^1] Today there are many companies manufacturing protein
chips using many types of techniques including spotting and gel methods.
The types of protein chips available include \"lab on a chip\", antibody
arrays and antigen arrays, as well as a wide range of chips containing
\"alternative capture agents\" such as proteins, substrates and nucleic
acids.

Analysis of protein chips comes with many challenges including dynamic
protein concentrations, the sheer number of proteins in a cell\'s
proteome, and the understanding of the probes for each protein. Steps
include the reading of the protein levels off the chip, and then the use
of computer software to analyze the massive amounts of data collected.

Applications of protein chip experiments include identifying biomarkers
for diseases, investigating protein-protein interactions, and testing
for the presence of antibodies in a sample. Protein chips have
applications in cancer research, medical diagnostics, homeland security
and proteomics.

This chapter will demonstrate why protein chips are changing the face of
proteomics, and why they will have an even larger impact in the future.

## History

### Nucleic Acid Microarrays

The use of microarrays for
gene expression profiling was first published in 1995.[^2] This
technology allowed scientists to analyze thousands of
mRNAs in a single experiment to
determine whether expression is different in disease states.
Unfortunately, mRNA levels within a cell are often poorly correlated
with actual protein abundance.[^3] This can be due to many factors
including degradation rate of mRNA versus proteins and
post-transcriptional controls and modifications. Measuring the amount of
protein directly would bypass any mRNA inconsistencies and give a true
level of gene function, however traditional protein characterization
methods were slow and cumbersome. These combined factors were the
impetus behind the creation of protein chips.

### Deficiency of Traditional Protein Characterization Methods

!A liquid chromatography / mass spectrometry (LC/MS) instrument. This
technique is low throughput compared to protein chips because protein
chips can test for thousands of proteins on a single chip in a single
experiment. instrument. This technique is low throughput compared to protein chips because protein chips can test for thousands of proteins on a single chip in a single experiment.")

Before the advent of protein chips, protein measuring and
characterization was done using two different methods: 2D gel
electrophoresis coupled with mass
spectrometry, and liquid
chromatography. These methods can
separate and visualize a large number of proteins per experiment,
however they are time consuming when compared to protein chips. Their
process is very low-throughput because of lack of automation.
Reproducibility is also a factor because of the large amount of sample
handling. A better, more standardized, higher-throughput method needed
to be invented for protein measuring and characterization.

### Protein Chip Precursors to Modern Day

!The equipment and reagents used in an Enzyme-linked Immunosorbent
Assay (ELISA), a precursor of protein
chips., a precursor of protein chips.")

Immunoassays, the precursor to protein chips available since the 1980s,
exploit the interactions between
antibodies and
antigens in order to detect their
concentrations in biological samples. Their creation, however, is
tedious and expensive. As a response to this, researchers at Harvard
University combined the technologies of immunoassays and DNA microarrays
to develop the protein chip.[^4] In their landmark paper, published in
2000, \"Printing Proteins as Microarrays for High-Throughput Function
Determination,\" Gavin MacBeath and Stuart Schreiber described how to
create protein chips and demonstrated three types of applications that
would benefit from this new technology. The strengths of their approach
were the use of readily available materials (i.e. glass slides,
polyacrylamide gel), the relative ease of implementation (robotic
microarray printers), and compatibility with standard instrumentation.

Within the past five years, many companies, including
Biacore,
Invitrogen, and
Sigma-Aldrich, have begun production of
industrial level protein array systems that can be used for drug
discovery and basic biological research. Commercial entities have made
protein chip research a streamlined and standardized process on the same
level as DNA microarrays compared to its inception in 2000.

Academic research plays a huge role in the development and improvement
of these technologies. The collaboration of academic research with
systems such as the Affymetrix GeneChip and the Human Genome Initiative
has allowed for friendly competition, resulting in the advancement of
technologies. With more develops come a better understanding and
encourages even more research towards these fields.

Affymetrix is a company that has been
manufactures microarrays, named GeneChip, since 1992. They have 13
locations across the world with headquarters located in the US
(California), UK, Japan, and China.[^5]

Next section:
Manufacture

## References

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<references/>
```
Chapter written by: Jonathan Keeling and Eric Foster\
Contact jwk3970@rit.edu, edf3480@rit.edu, ttl5439@rit.edu\

[^1]: MacBeath G, Schreiber S. (2000). Printing Proteins as Microarrays
    for High-Throughput Function Determination. Science. Sep 08; 289
    (5485): 1760-1764.

[^2]: Schena M, Shalon D, Davis RW, Brown PO. (1995). Quantitative
    monitoring of gene expression patterns with a complementary DNA
    microarray. Science. Oct 20; 270 (5235): 467-70.

[^3]: Gygi SP, Rochon Y, Franza B, Abersold R: Correlation between
    protein and mRNA abundance in yeast. Mol. Cell Biol. 19, 1720-1730
    (1999).

[^4]: MacBeath G, Schreiber S. (2000). Printing Proteins as Microarrays
    for High-Throughput Function Determination. Science. Sep 08; 289
    (5485): 1760-1764.

[^5]: \"Affymetrix.\" Wikipedia, The Free Encyclopedia. 5 Feb 2007,
    03:19 UTC. Wikimedia Foundation, Inc. Apr 2008
    \<<http://en.wikipedia.org/wiki/Affymetrix>\>


# Proteomics/Biomarkers

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### Mass spectrometry based targeted protein quantification: methods and applications

!Cross section of triple
Q{width="250"}

### Main Focus

The main focus of the paper was a review of the methods and applications
of using mass spectrometry to quantify proteins, especially those that
are in a concentration of less than µg/ml concentrations, in an attempt
to universalize a procedure.

### New terms

MALDI TOF/TOF: matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometer.

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Selected reaction monitoring (SRM): method in which a specific product ion from a specific parent ion is detected. All other ions with masses not specified in a predetermined range are filtered away leaving only ions with the mass in the range we are looking for. (source <http://en.wikipedia.org/wiki/Mass_chromatogram#Selected_reaction_monitoring_.28SRM.29>)

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Triple quadrupole: type of MS that contains a linear series of three quadrupoles. The first and third set act as mass filters, and the second is a collision cell. This type of MS can "filter" an ion of a known mass. (source <http://en.wikipedia.org/wiki/Quadrupole_mass_analyzer>)

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Hydrazide: class of organic compounds that share a common functional group characterized by a N-N covalent bond with one of the constituents being an acyl group. (source <http://en.wikipedia.org/wiki/Hydrazide>)

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Biomarker: biochemical feature that can be used to measure progress of the effects of treatment or a disease. (source <http://www.medterms.com/script/main/art.asp?articlekey=6685>)

For this summary, we will focus on protein biomarkers. Some diseases
which have protein biomarkers that show promise as a screening tool are
breast cancer, Alzheimer\'s, leukemia, ALS, and Parkinson\'s [^1]. A
series of six steps must be accomplished in order to successfully
validate a biomarker or set of biomakers: discovery, qualification,
verification, assay optimization, validation and commercialization[^2].
Once a biomarker is found and accepted, it can be used to possibly
predict and prevent the disease it\'s related to. The summary below
focuses on the quantification method of proteins in the search for and
identification of protein biomarkers. By finding ways in which to
universally quantify proteins, one can search for all biomarkers in one
screening rather than multiple screenings, once conclusive biomarkers
are identified.

### Summary

With the recent breakthroughs in technology, it is conceivable that is
possible to have a "universal" method or approach with minimal
restrictions to quantitatively assay a wide number of proteins in search
of potential biomarkers. Once a few potential biomarkers are discovered,
further research can be done to confirm or refute its use in clinical
applications. Another goal is to easily accumulate multiple detections
in a single measurement. Measurements are taken by identifying
synthetically stable isotopes attached to their respective proteins or
peptides. Each isotope mimics the peptide's endogenous counterparts
allowing high selectivity.

Mass spectrometry (MS) provides us with a powerful tool to compare two
different protein samples. It can be used for comparing the proteome of
a diseased sample against a normal sample at a global scale. This is
applied to a wide array of human diseases, with the hope that it will
lead to identification of biomarkers or even pathogenesis of a disease.
Traditionally, ELISA
(enzyme-linked immunosorbent assay) has been the major method for the
quantification of proteins with good sensitivity. Even today, it is the
"gold standard" for targeted protein quantification. The major drawback
with ELISA is the lack of availability of antibodies with high
specificity.

First attempts to determine the amount of specific proteins were done
using stable isotope dilution methods and MS approximately 20 years ago,
starting with atom bombardment MS and deuterium-labeled synthetic
polypeptides. Advances in MS instrumentation has increased our ability
to detect candidate proteins in complicated biological samples with high
sensitivity. To quantify the results, introduction of a stable isotope
(containing ^13^C or ^15^N, for example) to selected amino acids in a
reference peptide sequence provides a peptide with the same
physicochemical properties, that can be readily distinguished by MS from
the peptide in the target tissue or fluid. Studies have shown that
full-length proteins with stable isotopes can be used in quantification
of biomarkers in urine and water samples with nanomolar and picomolar
level sensitivities respectively.

There is a variety of MS platforms used for quantitative proteomics,
some of which are triple
quadrupole
(triple Q), matrix assisted laser desorption/ionization time-of-flight
tandem mass spectrometer (MALDI
TOF/TOF),
electrospray ionization (ESI) based on QTOF MS, and an ion trap
instrument using selective ion monitoring (SIM) mode. The most popular
of the platforms above is the triple Q. Demonstrations have shown that
it can multiplex and simultaneously target more than 50 peptides for
quantification in plasma in a single measurement. For targeted
quantitative analysis, coupling liquid chromatography with MALDI greatly
enhances the performance of MALDI bases MS. Some advantages of this
application include the ability of the techniques to be performed in
parallel with each other, it can be made a highly selective, data-driven
procedure, and the preservation of the sample to some degree for repeat
analysis. This technique is also highlighted by its potential high
throughput and excellent resolution.

One of the most important steps in quantification is sample preparation
which greatly influences sensitivity. One of the most common steps used
is the depletion of highly abundant proteins making it easier to enhance
analytical dynamic range and the detection of proteins in low
concentrations. One of the techniques performed is strong-cation
exchange chromatography (SCX) which has shown to give the ability to
detect peptides in the high pg/ml level, giving a 100-fold improvement
over direct plasma analysis.

Post-translational modification (PTM) is an important process to
understand since it is often involved in tumor progression, but can be a
problem to mimic due to the complexity and structure of the sugar chains
(as in glycosylation PTM). One experiment extracted N-linked
glycopeptides and de-glycosylated. This resulted in the conversion of
Asn to Asp and a difference in mass. This was utilized to make a
synthetic polypeptide to replicate an N-linked glycopeptide in its
glycosylated form.

Once of the main features of MS based quantification is for clinical
applications used to identify biomarkers associated with diseases. For
example, 177 protein candidates associated with stroke and
cardiovascular disease in plasma have been proposed. Some biomarkers
affiliated with stroke are S-100b, B-type neurotrophic growth factor,
von Willebrand factor and monocyte chemotactic protein-1[^3]. Other
biomarkers have been proposed to rheumatoid arthritis and breast cancer
among others.

One of the main goals of the ability to quantify proteins and peptides
is for personalized medicine. As technology advances, we will be able to
create techniques that easily assemble multiples detection in a single
measurement. Biomarkers from diseases can also be multiplexed in a
single assay, allowing us to possibly diagnose multiple diseases.
Ideally, a single-step preparation strategy is key, allowing for high
throughput and possible an automated process, reducing the amount of
human interaction and the chance of human error.

### Relevance to Proteomics Course

The ability to quantify proteins using mass spectrometry is a great tool
to compare a large number of proteins from a control sample with a test
sample in search of biomarkers. When a noticeable difference is
detected, further studies can be performed on those proteins. Major
breakthroughs in MS technology give us the capability to universally
approach developments to quantify a wide spectrum of proteins with
little restriction. It also gives us the ability to make more detections
per measurement. In the future, these approaches can give way to
personal medicine giving us the ability to screen individuals by
detecting multiple biomarkers from a single or multiple diseases.

### References

\[1\] Pharmaceutical Outsourcing Decisions. SPG Media Limited.
(http://www.pharmaceuticaloutsourcing.com/articles/pod003_014_power3.htm)

\[2\] Rifai, Nader, Gillette, Michael A., and Carr, Steven A. \"Protein
biomarker discovery and validation: the long and uncertain path to
clinical utility\". Nature Biotechnology 24, 971 - 983 (2006)
(http://www.nature.com/nbt/journal/v24/n8/abs/nbt1235.html)

\[3\] Reynolds, Mark A., et al. \"Early Biomarkers of Stroke\". Clinical
Chemistry 49 (2003): 1733-1739. Print.
(http://www.clinchem.org/cgi/content/abstract/49/10/1733)

[^1]:

[^2]:

[^3]:


# Proteomics/Experimental Protocols

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This page contains protocols that are frequently used in proteomics. You
are welcome to add protocols this chapter.

1.  /Plant Proteomics about Two Dimensional Gel
    Electrophoresis/