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Proteomics (Omics)

LECTURE 1 - INTRODUCTION AND THEORY

1. THE PROTEOME
Proteomics studies the set of proteins encoded by a genome that are expressed in a cell, tissue or
organism under specific conditions.
Of the proteome we can say:

• It is dynamic. It reflects, at the protein level, the changes in gene expression that underpin
processes such as cell differentiation and the response to environmental or environmental
changes. In other words, several proteomes can be generated from one genome,
depending on cell type, time of development or environmental conditions (pH, hypoxia,
exposure to drugs...).
• It is more diverse than the genome. In recent years, proteomics has broken the dogma of 1
gene = 1 protein, as it has been shown that one gene can generate more than one protein
or protein species with different properties.
The main sources of proteome variability are:
- At the DNA level. Genetic variation.
- At the RNA level. Post-transcriptional changes
(alternative splicing...).
- At the protein level. Post-translational
changes.

A numerical estimate of this reformulation for the human

genome is shown in the figure. From 20,000 genes, about
100,000 different transcripts have been found that encode
proteins that can be modified (i.e. PTMs) resulting in more than
one million protein species. Taking the genome as a starting
point, we observe an increase in complexity and diversity from
the genome to the transcriptome and from the transcriptome to the proteome.

It should be noted that:

- The cell does not express all possible proteins, which would be extremely expensive.
Therefore, there is a regulation of gene expression.
- There is no correlation between transcriptomics and proteomics.
- There are chemical modifications that can lead to proteins being eliminated.

2. BASIC TECHNIQUES OF PROTEOMICS

2.1. GENERAL STRATEGY OF ANALYSIS IN
PROTEOMICS
The general proteomics strategy is classified
depending on the molecule to be analysed by mass
spectrophotometry (MS).
• Top-down. Whole protein analysis.
• Botton-up. Analysis of small peptides
resulting from enzymatic digestion. This is
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the most commonly used strategy because it minimises complexity and increases
sample sensitivity.
2.2. PROCESSES OF THE PROTEOMIC ANALYSIS
1) Preparation of the protein extract. Please note:
• The method of extraction.
• Enrichment of a particular molecule.
i. Selective precipitation. Ex: Sulphuric acid allows precipitation of cellular
proteins, with the exception of histones.
ii. Differential centrifugation. Allows isolation of the proteome from different
organelles.
iii. Two-phase partitioning. Enables isolation of membrane proteins.
iv. Specific interactions. With affinity chromatography techniques we can use
a specific ligand to separate the protein from the rest of the proteome of
the cell (phosphoproteins, metalloproteins...).

• Modification of cysteine residues so that it cannot react with or alter the sample.
• Proteolytic digestion. Not in Top-down.
Ex: bottom-up, as protein fragmentation decreases sample complexity, enhances
sensitivity and improves mass accuracy.
Trypsin is the most commonly used protease. It cleaves the polypeptide chain after
Lys and Arg residues, provided the next residue is not a Pro. This leaves a positively
charged amino acid in each resulting peptide, thus promoting ionisation and
fragmentation.

2) Separation by:
• Electrophoretic techniques.
• Chromatographic techniques.

3) Mass spectrometry (MS). The final extract is passed through MS, which provides a large
amount of data, so we will require bioinformatics software for the analysis and

identification of the results.

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3. MASS SPECTROMETRY (MS)

Mass spectrometer (MS). An instrument that measures the mass-to-charge ratio of ions with high
accuracy. It consists of three parts:
• Ionisation source. Converts the analyte into gaseous ions which are accelerated by an
electric field towards the analyser. Types: Maldi, ESI
• Analyser. Separates ions according to their mass-to-charge ratio (m/z), using different
approaches. Types:
- TOF (Time of flight)
- Q (Quadroupole)
- Orbitrap
• Detector. It records the impact of individual ions, collecting the value of the mass-to-charge
ratio of each ion and generating the mass spectrum.

Combining the multiple options of ionisation source and analyser we obtain different techniques for
the analysis of biomolecules. Ex: MALDI-TOF (common in simple equipment), ESI-Q, etc.

PROTEIN MASS CALCULATION M = ((m/z) · z) − z

To calculate the mass of the protein, the m/z value is multiplied by the number of charges. The
number of charges is then subtracted to remove the weight of the incorporated protons. By having
multiple peaks of the same protein or peptide, the mass in each peak can be calculated, improving
the accuracy of the measurement.

3.1. ANALYZERS
Time Of Flight (TOF)
The time it takes for the ions to fly along the tube is proportional
to their mass-to-charge ratio.
The time of flight therefore depends on the potential energy with
which the ions enter the flight tube.
There is another variant of the TOF analyser called the reflectron.
1. Initial electric field. The reflectron incorporates a constant electric field at the end of the
flight tube which decreases the scattering of the ions due to energy differences during
acceleration. This results in narrower peaks in the spectrum.
2. Flying tube. The sample passes through the flight tube and the ion beam is reflected back
to the detector (positioned very close to the start of the tube).
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Quadrupole (Q)
The quadrupole consists of four parallel metal rollers through which the ions
travel. Two of the rollers are positively charged and two are negatively
charged.
When the ions are injected, a direct electric current is applied, causing them
to move towards the detector. At the same time, an oscillatory electric field
(RF) is applied, causing them to follow a wave-like trajectory.
Depending on the RF value, only ions of a certain m/z ratio can pass through the rollers. In this
way, the ions can be selected. To make a complete spectrum, it is necessary to make a sweep
of RF values (voltages).
The quadrupole can also be used as a collision cell if only AC current is applied (tandem MS). In
addition, the quadrupole is an inexpensive, compact analyser with good durability and reliability.

Orbitrap
The OrbiTrap consists of:
• Two concave electrodes
• A spindle-shaped electrode on the inside
The ions are injected tangentially from a device called a C-trap1 . Upon
entering the OrbiTrap, a potential is applied between the electrodes which
causes the ions to perform an orbital motion around the spindle. The
harmonic frequency of the motion is proportional to the m/z ratio. This
movement generates an image current in the detector, applying the Fourier
transform, the mass spectrum of all the ions that have been injected into the
OrbiTrap can be obtained.
3.1.1. PARAMETERS TO COMPARE THE ANALIZERS
The parameters for comparing the analysers are:
• Resolving power. It is determined using the half-peak amplitude. The mid-peak
amplitude indicates the minimum mass difference between 2 species to be detected
individually and corresponds directly to the resolution. Therefore, resolution and
resolving power should not be confused.
- The higher the resolving power, the better the analyser = narrower peaks.
- The lower the resolution, the better the analyser

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4. PEPTIDE MASS
FINGERPRINTING

Objective: to identify proteins.

A. A mixture of proteins is separated by 2D

electrophoresis.
B. Digestion and analysis. The resulting spots are
digested with a protease (trypsin) and analysed
by mass spectrometry (usually MALDI-TOF). The
result is a peptide mass list, which is used as an
imput in a bioinformatics search to identify the
proteins present in the sample.
C. Bioinformatics. Select a database and perform an in silico digestion of the
proteins in the database using the same protease. Then, the experimental
results are compared with the in silico digestion, only peptides with identical
mass or those within the estimated error range are selected.
D. Score calculation. A score is calculated from the number of matched peptides
to identify the proteins present. The data analysis of a peptide fingerprinting
experiment can be performed online.

5. TANDEM MS/MS
So far, all the applications mentioned above use simple single-analyser MS. However, there are
applications such as sequencing or determination of post-translational modifications that require
tandem mass spectrometry (MS/MS) because they require more than one analyser.

1) Ionisation. The sample is ionised.

2) Separation of the first analyser. The ionised sample is separated by its m/z ratio. At this
point the ions of interest (precursor ions) are obtained and we obtain a mass spectrum (MS1
).
3) Fragmentation. Precursor ions are fragmented and product ions are formed.
4) Separation in the second analyser. The product ions are separated in the second mass
analyser and we obtain the second mass spectrum (MS2 ).
a. The product ions can be refragmented, yielding additional spectra, MSn , where
n=number of mass spectra.
5) Detection.
MS/MS is used for peptide sequencing and identification of post-translational modifications and for
shotgun proteomics and
quantitative proteomics.

5.1. FRAGMENTATION
The polypeptide chain of a
protein can be broken at 3
different sites, generating
different product ions.

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• Ions a, b and c. Ions retaining at the N-terminus of the precursor ion.
• Ions x, y and z. Ions that retain the C-terminus of the precursor ion.
The predominance of one ion or the other depends on the fragmentation method chosen. For lower
energy fragmentation methods (CID/HCD), peptides fragment predominantly at amide bonds
(peptide bond). So we obtain b and y ions.

5.2. APPLICATIONS
PEPTIDE SEQUENCING
The procedure starts with peptide digestion, then ionisation and MS analysis, using a method known
as product-ion scanning.
1. 1st analyser. Selection of the desired ion in the first analyser.
2. Fragmentation. The desired ion is fragmented and the product ion is obtained.
3. 2nd analyser. The product ions are analysed in the second analyser on the basis of the mass
difference, which allows the sequence of the precursor ion to be determined.
This procedure can be repeated
several times until the complete
sequence is obtained.

IDENTIFICATION OF POST-
TRANSLATIONAL MODIFICATIONS (PTMs)
In order to carry out the study of peptide and protein PTMs, an enrichment step, specific to each
modification, is carried out first.
The identification of PTMs by MS is based on the difference in mass between the modified and
unmodified peptide. First the modified peptide is selected in the first analyser and after
fragmentation, the residue whose mass has changed corresponds to the modified residue.

• Neutral loss. For its detection, a synchronised scan of the two analysers is used, so that the mass
difference between the precursor and product ions remains constant and corresponds to the loss
of a neutral fragment. This scan allows the detection of specific functional groups, e.g. loss of
phosphorylation.

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LECTURE 3 - TARGETED, PTMs & QUANTITATIVE PROTEOMICS

Quantification can be:

• Relative comparison. The levels of a specific protein or all proteins in different samples are
compared. The proportion of change (increase, decrease or stay the same) of the proteins
between samples is obtained.
• Absolute quantification. Determination of the exact amount in mass units or concentration
of protein in a sample. It is used to quantify biomarkers.
Quantification methods can be classified according to:

• The type of quantification: relative or absolute

• Marking: whether to use it or not

1. TARGETED PROTEOMICS (SELECTIVE QUANTIFICATION)

Targeted proteomics aims at the absolute quantification of specific proteins. The simplest targeted
quantification method is called SRM (Selected Reaction Monitoring), based on the selection of so-
called proteotypic peptides. These have a sequence that is found exclusively in the protein of
interest in a particular species.
The chosen proteotypic peptide is selected in MS1 (usually of the quadrupole type) and fragmented.
We choose the product ions, generated in the second analyser, that have a higher intensity in the
MS2 spectrum.
The pair formed by a precursor ion and a product ion is called a transition. The SRM is usually
performed in a triple quadrupole.
i. 1st quadrupole. The precursor ion is selected.
ii. 2nd quadrupole. Collision is performed, as it acts
as a collision cell and not as an analyser.
iii. 3rd quadrupole. Select the product ions.
Several precursor ions can be selected allowing
quantification of more than one protein from the same
sample. This method is called MRN (Multiple Reaction Monitoring).

1.1. PRM (Parallel Reaction Monitoring)

The PRM has an OrbiTrap as a second analyser that allows it to detect all product ions originating
from a precursor ion at the same time. This technique allows absolute quantification by labelling
proteotypic peptides with heavy isotopes.

• SIS peptide. Proteotypic peptide labelled with heavy isotopes. Not all proteotypic peptides
are SIS peptides, to be SIS peptides they have to comply:
- Its size should range from 6 to 30 residues.
- They must not contain reactive amino acids such as Met or Cys, or PTMs.
- They must be the product of complete digestion, containing no undigested cut
sites.
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- High detectability in MS.

2. LABELED METHODS
Quantification methods with labelling are based on the incorporation of amino acids with heavy
isotopes (13C or 15N). The incorporation of heavy isotopes causes an increase in mass, which is
reflected in the m/z value and will depend on the number of isotopes added.
Experimentally, it has been shown that amino acids with heavy isotopes do not alter the chemical
properties or chromatographic behaviour of peptides and proteins. For that reason, a normal
peptide and its heavy analogue are kept together during sample processing and separation prior to
MS.
Isotopic labelling can also be labelling:
• Metabolic. If isotopes are incorporated during cell culture or growth.
• Isobaric. If reagents with the same mass are used to mark multiple samples.

2.1. SILAC (Stable isotope labelling by amino acids in cell culture)

The SILAC method is based on the incorporation of labelled amino acids as proteins are synthesised
in the cell. It is therefore a metabolic and isotopic labelling method.
It works under two conditions:
• Control condition. Proteins are not modified
• Condition flagged. A drug or treatment has been added to assess its effects on the
proteome. A labelled amino acid has been added to the culture medium of the labelled
sample for sufficient time to be incorporated into the proteins.
After the incubation time (48-72 hours), the extract is prepared for each condition. The extracts are
mixed in equal amounts and if necessary, fractionation can be performed, followed by digestion and
finally MS analysis.
The mass spectrum obtained shows the same peptide in its light and heavy form. The difference in
mass is determined by the number of heavy isotopes incorporated and the number of labelled amino
acids. The ratio between the intensities of the two peptides allows a relative quantification of the
changes in the proteome under the tested conditions.

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ABSOLUTE SILAC QUANTIFICATION SILAC (Absolute SILAC)

Method based on the addition of pre-tagged proteins to a sample.
1. Tagging. Tagging of the protein can be done by expressing it as a recombinant protein in
bacteria.
2. Purification and quantification. After protein purification, the protein is accurately
quantified and known concentrations can be added to the biological sample to be analysed.
3. Mass spectra. This will show peaks of the labelled protein in the mass spectrum and absolute
quantification of the protein of interest can be performed by comparing the areas of the
extracted chromatogram (XIC).
Absolute quantification will be very accurate for the labelled protein. This protein can also be taken
as an internal standard to estimate the amount of other proteins or to perform a relative
quantification for the rest.

3. LABELLED METHODS FOR MORE THAN 2 SAMPLES: ISOBARIC LABELLING

The SILAC method allowed the comparison of two samples. However, by means of isobaric labelling
(using a group of molecules of the same molecular weight, isobaric) we can compare more than two
samples in the same mass spectrum. The TMT method stands out.

3.1. TMT (Tandem Mass Tag)

TMT is another isobaric method, but in this case, the isobaric (isotopically labelled) reagents are:
• Reporter group. Which is released after fragmentation (i.e. by collision).
• Mass normaliser. Similar performance to the iTRAQ balance group.
• Reactive group. It can be of three types:
- Reaction with primary amines.
- Reaction with cysteines.
- Reaction with the carbonyl group.
This method, like iTRAQ, allows multiplexing. The reporter ions have known masses, approximately
6 mDa apart, so very precise analysers (such as the OrbiTrap) are required.
The experimental strategy of the TMT is similar to that of iTRAQ, but in this case the reagent sets are
of 10 and 16 samples, so it is also widely used in clinical analysis. It
has a higher throughput capacity than iTRAQ.