Welcome to the MOOCs NPTEL Course on Mass
Spectrometry based Proteomics.
The proteome describes the protein compliment
expressed by a genome.
The extent of diversity and complexity due
to alternative splicing and post translational
modifications is tremendous.
Therefore, studying proteins and proteome
becomes very important.
In recent years, mass spectrometry has played
a major role in Proteomics level investigation.
And in 2014, 2 human proteome reference maps
were published using high resolution mass
spectrometry.
This course is divided into 4 modules.
Each module, we will be finishing in 1 week.
There will be 20 lectures of around 30 minutes
duration which will cover the key concepts,
experiments and laboratory demonstrations
to explain the concept effectively to the
students.
The first module will discuss about the basic
concepts of proteomics and the importance
of sample preparation.
Further we will discuss the fundamentals of
mass spectrometry and the advancements in
technology.
Lecture 1 will focus on basic understanding
of proteomics.
The technological advancementsů.
ůin analytical techniques with increased
sensitivity, ů
ů resolution, ů
...and capability to carry out high throughput
studiesů
ů have led to the transition fromů
ů protein chemistryů
ů to the new field of proteomics.
How limitations of mass spectrometry for protein
analysis ů
ů. was overcome by development of soft ionization
techniquesů
ůsuch as MALDI and ESI will be discussed
in this lecture
In lecture 2, we will talk about sample preparations
for proteomics applications.
Protein extraction is a crucial step for any
proteomic investigation.
The protein extraction method involves cell
lysis, prevention of proteolysis during lysis,
different types of protein precipitation methods,
ů
ůprotein solubilization, ů
ůand removal of ů
ůvarious interfering components.
The protocol standardization becomes a major
challenge in proteomics ů
ůand there is no generic protocolů
ůwhich exists in literature ů
ů. which can be used ů
ů for every sample type.
Therefore, we will discuss a few sample preparation
strategiesů
ůand how a good sampleů
ůcan be prepared ů
ů. for the proteomic analysis.
We will continue the protocol standardizationů
ů using bacteria and discuss about cell lysis,
ů
ů. protein precipitation and quantification
and discuss about trizol extraction protocol.
In lecture 4, we will talk about in-gel digestion
or in-solution digestion of the protein which
is essential prior to the mass spectrometry
analysis.
Often the in-gel digestion is used to extract
proteinsů
or peptidesů
ů separated on the gel electrophoresis.
In gel proteolytic digestion,
ůthe in-gel proteolytic digestion is performed
ů
ů. to cleave the protein of interest present
within the polyacrylamide matrix.
Mass spectrometric identification of the target
protein greatly depends onů
ů the efficacy of the in-gel digestion protocol
which generates mixture of peptides from the
target protein through proteolytic digestion.
In lecture 5ů
ůwe will talk ů
ů about ů
ůthe fundamentals of mass spectrometry.
Mass spec is highly sensitive balance to measure
the mass of the molecule in vacuum based on
mass to charge ratio.
It consists of an ionization source to ionize
the molecule, a mass analyzer to resolve the
analyte molecules in vacuum, and a detector
to read the signals coming from the mass analyzers.
There are different ionization sources and
mass analyzers being used for the proteomic
analysis.
If you know the principle of each one of these,
it becomes much easier to select which configuration
to use and when for your proteomic investigations.
In the second week, we will discuss in detail
about different types of chromatographic techniquesů
ů like gel filtration, ion exchange chromatography,
ů
ůaffinity chromatography, SCX or Strong cation
exchangeů
ůand reverse phase chromatography.
As we will progressů
ů through lectures 6 and 7, we will discussů
ů how different chromatographic techniques
work based on different principlesů
ůand how one could employ those ů
ůin the proteomics workflow.
Lecture 8 will focus onů
ůionization sources.
The ionization sources are responsible forů
ůconverting the analyte molecule into ů
ůgas phase ions in vacuum.
The ions generated by the ionization source
ů
ůare then integrated with the mass analyzer.
. The commonly used ů
ů ionization sources are MALDI ů
ůand ESI.
In lecture 9,
ůwe will talk about mass analyzers.
The mass analyzersů
ůresolve the ions produced by ionization
source ů
ůon the basis of ů
ů their mass to charge ratio.
Various characteristics such as resolving
powerů
ůaccuracy, mass range and speed determine
theů
ůefficiency of the analyzers.
The commonly used mass analyzers are ů
ůTime of Flight TOF, Quadrupoles and Ion
Traps.
The sample preparation strategy prior to mass
spec analysis ů
ůwill be discussedů
ůin lecture 10
Onceů
ůthe protein sample has been digested, all
the salts, buffers and any detergents ů
.. must be removed from the sample which can
be effectively performed ů
ůby using some filters ů
ů such as ZipTips.
It offers several ů
ů advantages such as pick purificationů
ů sample enrichment and ensures that there
is no contamination.
However, it can purify only a limited volume
of the sample and ů
ůalso adsorbs some amount of protein sample
ů
ů. thereby leading to losses.
Further we will talk about how to use MALDI
to analyze your samples in high throughput
manner.
The third module will cover introduction to
quantitative proteomics and hybrid mass spec
configurations.
The complexity and dynamic nature of proteomes
presents major technological challenges.
Mass spectrometry advancements have improved
in high throughput identification and quantification
of proteins and now offer an opportunity to
understand human diseases and discover biomarkers.
The lectures 11 and 12 will cover hybrid MS/MS
configurationsů
ůas well as ů
ůdiscussion on two latest hybrid MS technologies,
ů
... Q-TOF and Orbitrap.
The basics of quantitative proteomic analysis
ů
and what are the different types of ů
ůquantitative methods that existů
ů in literature using mass spectrometry will
be discussed.
In lecture 13, we will talk about quantitative
proteomic analysis using Stable Isotope Labeling
by Amino acids in Cell culture or SILAC.
SILAC is an in vivo labeling method; the labels
can be introduced in vivo by growing an organism
in the media enriched with specific isotopes.
There are different ways of in vivo labeling
such as enrichment of 15 Nitrogen media, Culture
Derived Isotope Tags known as CDIT or SILAC.
In this lecture, the major emphasis will be
on the SILAC method for quantitative proteomic
analysis.
The next lecture will talk about quantitative
proteomics using iTRAQ technique.
iTRAQ involves identification and quantification
of ů
ů complex protein mixtures by MS based quantitative
proteomic techniques.
The iTRAQ reagent consists of amine specific
stable isotope reagents which can label peptides
of up to 4 or different biological samples.
The iTRAQ method provides multiplexing capabilities
of 4 or 8 sample analysis which is not possible
ů
ů. using iCAT, where only 2 samples ů
ůcan be labeled and analyzed.
In ITRAQ, 4 plates reagent setsů
ůthe m/z value ranging ů.
ůfrom 114 to 117.
There is a balance groupů
ůof mass 28 to 31 Dalton, therefore the overall
mass of reporter and balancer component ů
ů remains constant.
During the MS/MS fragmentation, the reporter
ions give the peaks at 114, 15, 16, and 17ů
ů which provides the information about ů
ů the peptide or protein abundance.
The next lecture will focus on another quantitative
proteomic technique based on Tandem Mass Tags
known as TMT.
TMT is also in-vitro labeling method which
is similar to the iTRAQ method.
TMT is a MS/MS based quantitative technique
which uses the isotope labeled model referred
as Tandem Mass Tags.
It provides the accurate quantification of
peptides and proteins.
By using different types of TMT tags, one
could perform multiplexing experiments of
2, 4, 6 or 10 plex.
In the last module, we will talk about quantitative
proteomic data analysis and some aspects of
System Biology applications.
The quantitative proteomic technologies aim
to identify the differentially expressed proteins
in a biological sample.
The differential expression of proteins can
be caused by a diseased state or various external
factors like stress, drugs or different experimental
conditions.
The data analysis is ů
ůan important step for protein identification
and quantification in proteomics workflow.
The accurate quantification of protein abundance
becomes very important for the quantitative
proteomic analysis.
This lecture will focus on data analysis for
the protein identification using ů
ů Mascot and protein quantification using
iTRAQ-based workflow.
The next 2 lectures, 17 and 18, will cover
proteomics and Systems Biology.
So, what is Systems Biology?
The Systems Biology is the examination of
the biological entity as an integrated system
rather than studying its individual characteristics,
reactions and components.
And that is what is termed as Systems Biology.
The distinct approaches of Systems Biology
include the model-based and data-based methods.
The model-based approach involves some prior
information which can be implemented in these
models, where as in the data-based methodology,
the objective is to find the new phenomena.
Some of these details will be covered in these
lectures on Systems Biology.
Proteomics has a wide range of applications,
from understanding the physiology of micro-organisms
to biomarker discovery for cancer and other
diseases.
Towards the end, I would like to cover proteomics
applications and challenges.
The last lecture will focus on proteomics
challenges.
Mass spectrometry is one of the best inventions
in the proteomics field in recent years.
It has been able to achieve many milestones
including the draft human proteome maps.
Though Mass Spec is a sophisticated instrument
and provides very high throughput robust capability
of analyzing proteome, still it has many challenges
to overcome in the future.
The post translational modification analysis
using Mass Spec is one of the challenges.
Additionally, the dynamic range of proteins,
inadequate coverage of whole proteome and
accuracy of quantification are challenging
in this field.
Overall, this course will provide the basic
knowledge of mass spectrometry with focus
on quantitative proteomics.
Hope these concepts and understanding will
be useful for your research.
Thank you.
