proteomic study typically focuses on one or more of the following aspects of a target organism’s proteome at a time to

slowly build on existing knowledge:

Protein Which proteins are normally expressed in a particular cell type, tissue or organism as a whole, or which proteins are
identification differentially expressed?

Protein Measures total (“steady-state”) protein abundance, as well as investigating the rate of protein turnover (i.e., how
quantification quickly proteins cycle between being produced and undergoing degradation).

Where a protein is expressed and/or accumulates is just as crucial to protein function as the timing of expression, as
Protein localization cellular localization controls which molecular interaction partners and targets are available.

Post-translational modifications can affect protein activation, localization, stability, interactions and signal transduction
Post-translational among other protein characteristics, thereby adding a significant layer of biological complexity.
modifications

Functional This area of proteomics is focused on identifying the biological functions of specific individual proteins, classes of
proteomics proteins (e.g., kinases) or whole protein interaction networks.

Structural studies yield important insights into protein function, the “druggability” of protein targets for drug discovery,
Structural proteom and drug design.
ics
Protein-protein
interactions Investigates how proteins interact with each other, which proteins interact, and when and where they interact.
Proteomics: Techniques

• Low-throughput methods:

1. Antibody-based methods
2. Gel-based methods
3. Chromatography-based methods

• High-throughput methods:

1. Analytical, functional and reverse-phase microarrays

2. Mass spectrometry-based proteomics
SDS binds to proteins at approximately 1.4 grams of SDS per gram of protein, coating the polypeptide chains and
linearizing them. This binding imparts a uniform negative charge to the proteins, ensuring that the electrophoretic
mobility is determined solely by size, as the charge-to-mass ratio is constant.
 Preproinsulin: 12 kilo daltons (kDa)
Proinsulin: 9.4 kDa
Insulin: 5.8 kDa

Human insulin is a peptide hormone made up of 51 amino acids,

including 21 amino acids in the A-chain and 30 amino acids in the B-
chain. The A-chain and B-chain are linked by two disulfide bonds.
 Second dimension: SDS-PAGE

https://www.creative-proteomics.com/blog/index.php/two-dimensional-gel-electrophoresis-2-de/
Liquid chromatography (LC)
LC is a technique used to separate and analyze components of a mixture:

A liquid mobile phase carries a sample through a stationary phase, which is made up of small particles. The components of the
sample interact with the stationary phase in different ways, based on their size, adsorption, ion-exchange, or partitioning.
Size Exclusion Chromatography (SEC):
Mass Spectrometry
• Introduction
• Principle of Mass Spectrometry (MS)
• Basics
• Ionization - MALDI and ESI
• Mass analyser – QqQ, TOF, Q-TOF, Ion Trap
• Sample Preparation
• Sample Analysis: Quantitative and Qualitative
 Mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to
measure the mass-to-charge ratio of ions. The results are
presented as a mass spectrum, a plot of intensity as a function of
the mass-to-charge ratio.

Mass spectrometry is an important method for the accurate mass

determination and characterization of proteins
Mass-spectrometers: A century old
legacy

• 1886 Eugen Goldstein, Gas discharge of in cathode channel called it Anode

ray
•Wilhelm Wein used magnetic and electric field - charge-to-mass ratio (q/m)
•The history of MS began with Sir J. J. Thomson of the Cavendish Laboratory.
•The Calutron, a three-story-high version of Nier's sector instrument,
separated uranium-235 for the first atomic bomb.
Evolution of Mass-
spectrometry

Sir J.J Thomson

John B. Fenn Nobel Prize in
Nobel Prize in Physics, 1906
Chemistry, 2002

Wolfgang Paul
Nobel Prize in Physics,
1989
Francis Wiliam
Koichi Tanaka Aston
Nobel Prize in Nobel Prize in
Chemistry, 2002 Chemistry, 1922
Principle
• MS converts the components (peptides/ proteins) to ions and then
analyses them on the basis of their mass/charge (m/z).
• Process occurs in gaseous and vacuum
1. Source: produces ions from the sample which gives ions defined
energy or velocity.
2. Mass analyser: Resolves ions based on their m/z ratio
3. Detector: Detects ion and assigns the mass
4. Data collection: Records the data and used interpretation with the
aid of PCs and software/s.
Here are some types of peaks in a mass spectrum:
Base peak
The peak with the highest intensity, which is assigned a relative
intensity of 100%.

Molecular ion peak

Also called the parent peak, this peak corresponds to the
compound's molecular ion. The m/z ratio of this peak directly
corresponds to the compound's molecular weight.

M+1 peak
A smaller peak that corresponds to an isotope in the sample.

M+2 peak
Another small peak that corresponds to isotopes of certain
elements, such as chlorine and oxygen.

Metastable peaks
Broad peaks with low intensity at non-integer mass values.
These peaks are caused by ions that have shorter lifetimes than
the time it takes to travel from the ionization chamber to the
detector
Spectrum of CO2
In protein mass spectrometry, peaks in the mass spectrum correspond to different parts
of a protein molecule. The mass-to-charge ratio (m/z) of each peak defines which part of
the protein it corresponds to. The m/z ratio is based on the charges carried by each part
of the protein. The more charges a part of the protein has, the smaller its m/z ratio, and
the farther left it will appear on the spectrum.
Basic Components
 Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
(MALDI-MS)
MALDI is a soft ionization technique
used in mass spectrometry (MS) that
produces rapid and efficient ionization
of a wide variety of molecules

The sample for MALDI is uniformly mixed in a large quantity of matrix. The matrix absorbs the ultraviolet light (nitrogen laser
light, wavelength 337 nm) and converts it to heat energy.
In a typical MS procedure, a sample, which may be solid, liquid, or gaseous, is ionized, for example by bombarding it with
a beam of electrons. This may cause some of the sample's molecules to break up into positively charged fragments or
simply become positively charged without fragmenting. These ions (fragments) are then separated according to their
mass-to-charge ratio, for example by accelerating them and subjecting them to an electric or magnetic field: ions of the
same mass-to-charge ratio will undergo the same amount of deflection.[1] The ions are detected by a mechanism
capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the signal
intensity of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be
identified by correlating known masses (e.g. an entire molecule) to the identified masses or through a characteristic
fragmentation pattern.
Ion Sources
• Gas Phase Ionization:
Electron Impact (EI)
Chemical Ionization (CI)
• Desorption Ionization:
Cf Plasma Desorption (PDMS)
252

Fast Atom Bombardment (FAB) / Secondary Ion MS (SIMS)

Laser Desorption (LDMS)
Matrix Assisted Laser Desorption (MALDI)
• Spray Ionization:
Thermospray (TSP)
Atmospheric Pressure Chemical Ionization (APCI)
Electrospray (atmospheric pressure ionization) (ESI, API)
 Methods and approaches
• electrospray ionization (ESI) : In electrospray, the ions are
created from proteins in solution, and it allows fragile
molecules to be ionized intact, sometimes preserving non-
covalent interactions.
• matrix-assisted laser desorption/ionization (MALDI). In
MALDI, the proteins are embedded within a matrix
normally in a solid form, and ions are created by pulses of
laser light.
Electrospray produces more multiply-charged ions than MALDI, allowing for measurement of high mass protein and
better fragmentation for identification, while MALDI is fast and less likely to be affected by contaminants, buffers and
additives.
The most widely used instrument for peptide mass analysis are the MALDI-TOF instruments as they permit the
acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec).
Electrospray Ionization [ESI]
• The Liquid Chromatography (LC) eluent is sprayed (nebulized) into a
spray chamber at atmospheric pressure in the presence of a strong
electrostatic field and heated drying gas.
• Four steps process
• Ion formation – in atmospheric pressure ionization
• Nebulization - aerosol generation
• Desolvation - heated drying gas evaporates the solvent
• Ion evaporation
Electrospray Ionization
MALDI Vs ESI
MALDI ESI
• Admixture of sample & matrix • Sample in solution
• MS spectra (parent ions) and MS-MS
• Sample is spotted on slide (product ions)is available
• Peptide ions masses are generated • Useful for sequence determination and
• Peptide mass fingerprint is used for matched with the database for best fit
protein analysis
identification of proteins
• 3 amino acids to high Mw can be studied
• High Mw mass remains challenge
• PTM with very low difference can be
• Sequence of peptides cannot be analysed
determined • Discovery and Quantitative proteomics
• PTM detection is difficult • Very expensive
Mass Analysers
• Quadropole (Triple quadropole or QQQ)
• Ion Trap
• Time of flight (ToF)
• Fourier Transform Ion Cyclotron Resonance
Different Mass analyser

Adopted from: Aebersoldand Mann, 2003)

Time-of-flight mass spectrometry (TOF-MS) is a mass spectrometry
technique that measures the mass-to-charge ratio of ions by
measuring how long it takes them to travel a set distance.
 Fourier transform ion cyclotron resonance (FT-ICR).: Fourier-transform ion
cyclotron resonance mass spectrometry is a type of mass analyzer for
determining the mass-to-charge ratio of ions based on the cyclotron
frequency of the ions in a fixed magnetic field.
Experimental design
• Objective of study for MS
• Determination of intact mass
• Structural proteomics
• Discovery proteomics
• Quantitative proteomics
• Post translation modifications
• Functional proteomics
• Targeted /untargeted proteomics
• Glycomics
• Metabolomics
• Lipidomics
Tandem mass spectrometry (MS/MS):Tandem mass spectrometry
(MS/MS) is used to measure fragmentation spectra and identify
proteins at high speed and accuracy. Collision-induced dissociation
is used in mainstream applications to generate a set of fragments
from a specific peptide ion. The fragmentation process primarily
gives rise to cleavage products that break along peptide bonds.
1. Lysate preparation. Lysis, fractionation, depletion, enrichment, and dialysis.

2. In-solution or in-gel digestion.

a. Protein denaturation using chaotropic agents such as urea and guanidine;

b. Reduction of disulfide bridges using DTT;

c. Alkylation of the cysteines by iodoacetic acid or iodoacetamide;

d. Remove regents and exchange buffer;

e. Overnight denaturation with trypsin or other proteases in an ammonium bicarbonate buffer at suitable pH and
temperature for about 18 h;

f. Stop the digestion by the addition of (formic) acid.

3. Protein enrichment/cleanup.

We also provide the high-throughput, automated protein digestion service with 96-well formats.
Protein Digestion

In-Gel Digestion: This method involves digesting proteins directly within a

polyacrylamide gel matrix. It is ideal for proteins separated by 1-D or 2-D
electrophoresis. In-gel digestion is known for its robustness, effectiveness, and
reproducibility. Despite these advantages, it is a labor-intensive and time-
consuming process.

In-Solution Digestion: This approach starts with protein precipitation using

chloroform/methanol, followed by resolubilization in urea and subsequent
digestion with trypsin or other proteases. This method is generally quicker than in-
gel digestion but may result in some sample loss due to incomplete resolubilization
of aggregated proteins.
Protein digestion techniques
Why digest proteins?

• Greater the mass of protein higher the error rate in finding the ptn
mass

• Not all proteins are amenable to intact (whole) mass measurement

• Sensitivity of peptide mass measurement is better than protein mass

measurement
Protein digestion
• Should cleave protein at specific sites

• Peptide of length 6-20 – ideal for MS analysis

• Objective: to produce high yield of peps of optimal length for MS

analysis
Proteases
• 1000s of distinct protease available

• Stable, well characterized enzyme, well defined

specificity for cleavage

• Can be easily obtained in high quantity and high purity

• Undertake many task for ptn remodeling – essential for

higher organism
Protein Digestion
Prior step
• Denaturation- dissociates and unfolds proteins
• Chaotropes: Urea, Guanidine

• Disulfide Bond Cleavage

• Two Common Reducing Agents: Dithiothreitol, beta-mercaptoethanol

• Note pH dependence

• Cysteine Alkylation- prevents reoxidation to form disulfides (Cysteine

scrambles)
• Two common Alkylating Agents: Iodoacetamide, Vinylpyridine
Protein Digestion
An essential step for mass spec analysis
• Chemical Methods:
• Acid Hydrolysis, various [H+], time, temperature
• Cyanogen Bromide cleavage, C-term to Methionine gives a homoserine lactone at
Met
• Enzymes:
• Trypsin C-term to Lys, Arg pH 8.5
• Chymotrypsin C-term to Y, F, W, H, L pH 8.5
• S. aureus V8 protease C-term to Glu pH 8
• S. aureus V8 protease C-term to Glu, Asp pH 5
• Achromobacter protease (Lys-C) C-term to Lys pH 8
• Arg-C C-term to Arg pH 8
• Asp-N N-term to Asp pH 8
• Thermolysin N-term to L, I, M, F, W pH 8.5
• PNGase F N-glycans pH 6
• The specificities of proteases is a very useful tool to bear in mind.
• Specificity of these methods is variable: some excellent, some almost
none
Protein Digestion for Mass Spectrometry
Conventional Digestion
Protocol
Briefly, 60 μg of protein in 60 μL of PBS buffer
was evaporated and resuspended in 6 μL of 8
M urea in water containing 10 mM DTT.

This solution was incubated for 1 h at 37 °C.

Iodoacetamide was added to a final
concentration of 55 mM, the mixture was
incubated for 45 min in the dark, DTT was
then added to final concentration of 65 mM,
and this mixture was incubated for 1 h. The
solution was then diluted with 20 mM
NH4HCO3 pH 8.0 to a final concentration of 1 Iodoacetamide (IAA) is an organic compound that's used as an alkylating
M urea. agent to study proteins:

Trypsin (2 μg) was added to 60 μL of diluted How it works

denatured protein, and this was incubated at IAA reacts with the free sulfhydryl groups of cysteine residues in proteins,
37 °C overnight. Digested samples were used forming covalent bonds that prevent the protein from forming disulfide
without further purification by mixing with bonds.
matrix and spotting on MALDI plates.
Protein Digestion at Different Temperatures
In-solution digestion at elevated
temperatures was done without
denaturation or alkylation of proteins. In
each experiment 1 μL (1 μg) of protein in
water or PBS buffer, 1μL (140 ng) of trypsin
in water and 1 μL of DTT (1.5 mM) in 100
mM NH4HCO3 pH 8.0 were loaded into
TempAssure PCR tubes, mixed, placed in the
thermocycler, and incubated for 20 s at the
desired temperature.

Optimum temperature of trypsin and chymotrypsin in the

hydrolysis of whey proteins at pH 9.0. Substrate concentration:
40.0 gL-1 .
Protein Digestion: Dependence
on pH, DTT, NaCl

For the experiments with DTT, PCR tubes were

prepared with 1 μL (1 μg) of protein in water or PBS
buffer, 1 μL (140 ng) of trypsin in water, and 1 μL of
DTT in 100 mM NH4HCO3, pH 8.0. Different
concentrations of DTT were added to make final
concentrations of 0, 0.25, 0.5, 1.0, 2.5, 5.0 or 10 mM.
For the experiments with salt, PCR tubes contained 1
μL (1 μg) of protein in water, 1 μL (140 ng) of trypsin
in water, 1 μL of solution containing DTT (1.5 mM) in
100 mM NH4HCO3 and different amounts of 4 M
NaCl was added to get final concentrations of NaCl of
100 or 1000 mM. Samples were prepared and kept on
ice until spotted. At least 3 replicas were used for
each experiment.
Thermocycler Digestion Method

Proteins were digested in solution with trypsin, without chemical denaturation. In each experiment, 1 μL (1
μg) of protein in water or PBS buffer, 1 μL of 1.5 mM DTT in 100 mM NH4HCO3 and 1 μL of trypsin in water
(1.5 μg, final concentration of 21 μM) were loaded into TempAssure PCR tubes and mixed. The digestion
reaction was performed in the thermocycler, using a program with 20 s at the optimal digestion
temperature for the protein, or for unknowns and difficult to digest proteins, with 20 s at each of the
following temperatures: 49, 50, 51, 52, 53, 54, and 55 °C.
Trypsin In-gel
digestion

Displays good activity both

in solution and in-gel
In-Gel digestions

Band/spot of interest is cut from gel

Destained

Treated with protease(trypsin)

Enzyme penetrates the gel matrix and cleaves digests the

protein to peps

Eluted from gel by washing

Serine proteases (or serine endopeptidases) are enzymes that cleave peptide bonds in proteins. Serine
serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both
eukaryotes and prokaryotes.

Cysteine proteases, also known as thiol proteases, are enzymes that degrade proteins (Verma et al., 2016).
These enzymes share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a
catalytic triad or dyad set of organization

Aspartic proteases (also "aspartyl proteases", "aspartic endopeptidases") are a catalytic type of protease
enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of
their peptide substrates.

Metalloproteases are a class of enzymes that use a metal ion, usually zinc or cobalt, as part of their
catalytic mechanism. collegenase and gelatinase

Aminopeptidase
Aminopeptidases are enzymes that catalyze the cleavage of amino acids from the amino terminus (N-
terminus) of proteins or peptides (exopeptidases).

Carboxypeptidase is a protease enzyme that hydrolyzes (cleaves) a peptide bond at the carboxy-terminal
(C-terminal) end of a protein or peptide.
• Chymotrypsin is a digestive enzyme that cleaves peptide
bonds in proteins at the C-terminal side of amide linkages
that contain aromatic amino acids, such as tyrosine,
phenylalanine, or tryptophan:
Glu-C
• Endo-proteinase

• Cleaves at the carboxyl side of glutamate in ammonium

bicarbonate/acetate buffer

• Can be used for in-gel digestion also

Other proteases

• Lys-c, chymotrypsin, Asp-N

• Specificity – not ideal

• Chymotrypsin – cleaves frequently(at Y,F,W) - yields too many small

peps

• Others – yields fewer, larger peps.

Other non specific proteases
• Subtilysin, pepsin, proteinase K and pronase

• Lacks specificity; Random cleavage

• Produces multiple overlapping peptides

Chemical cleavage
• Cyanogen bromide (CNBr) – cleaves at methionine
residues

• Frequency of Met is less- yields fewer, larger fragments

• Larger fragments – not suitable for MS analysis

In solution and in gel digestion workflow for MS
analysis