# 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)

