Submitted To:- Dr.

Mohibullah Shah

Topic:- Targeted Proteomics

Presented by:- Kainat Bilal

PHD-BC-20-01
 Contents
• Introduction
• Global vs Targeted Proteomics
• Methods used in Targeted Proteomics
• General Procedure
• Protein Quantification
• Applications
• Summary of some Targeted Proteomic Studies
• Conclusion
 Proteomics relies on three basic technological
cornerstones that include;

• A method to fractionate complex protein or

peptide mixtures

• MS to acquire the data necessary to identify

individual proteins

• Bioinformatics to analyze and assemble the MS

data
 Protein Source

Protein Extraction

Fractionation

Identification

Biological Analysis (Bioinformatics)

 MS based proteomics approach can be either global
(shotgun) or targeted.
Shotgun/Global proteomics:
-Indiscriminately identifies most abundant proteins

-No prior knowledge required for protein detection

-Information obtained for a large number of proteins

Targeted proteomics:
-Selectively targets one protein or a set of proteins

-Requires prior knowledge of protein mass and sequence

-Limited number of proteins are assayed at once

• Global proteomics provides the key information required to build
subsequent targeted experiments.

• This information includes identity of protein, amino acid sequence,

retention time and charge state of peptides, and the distribution and
intensity of the fragment ions in the MS/MS spectrum.

• Targeted experiments are primarily performed on a triple-quadrupole

(QQQ) and hybrid quadrupole-linear ion trap (QTrap) mass
spectrometers using a data acquisition method known as Selected
Reaction Monitoring (SRM).

• Targeted experiments known as Parallel Reaction Monitoring (PRM)

are performed on hybrid quadrupole-Orbitrap (q-OT).
Methods used in Targeted
Proteomics
• Selected/Single Reaction Monitoring (SRM)

• Multiple Reaction Monitoring (MRM)

• Parallel Reaction Monitoring (PRM)

 Overview
• SRM monitors only a single fixed mass window while
MRM scans rapidly over multiple (very narrow) mass
windows and predefined transitions are measured
sequentially.

• MRM is an application of SRM to multiple product ions

from one or more precursor ions.

• PRM performs a full scan of each transition by a

precursor ion, that is, parallel monitoring  of all
fragments from the precursor ion.
 SRM/MRM
An SRM experiment is generally performed in a triple quadrupole
(QqQ) mass spectrometer. Triple quads contain 3 quadrupoles in
series that are programed to selectively stabilize your ion of interest.
Quadrupoles act as a mass filter.
 SRM/MRM analysis uses 2 stages of mass
filtering
• Q1: Peptide precursor is selected (parent/precursor
ions) that are identical to the pre-set mass to charge
ratio.
• q2: peptide is fragmented via collision induced
dissociation.
• Q3: Peptide fragment is selected (fragment ion).
• The specific pair of m/z values associated with the
precursor and fragment ions selected is referred to as
a ‘transition’.
• Usually ≥ 3 transitions are monitored for each peptide
of interest.
 Parallel  reaction monitoring (PRM) 

PRM is based on Q-Orbitrap as the representative quadrupole-high

resolution mass spectrum  platform. Unlike the SRM, which performs
one transition at a time, the PRM performs  a full scan of each
transition by a precursor ion, that is, parallel monitoring  of all
fragments from the precursor ion.
• The principle of this technique is comparable to SRM/MRM, but it  is
more convenient in assay development for absolute quantification of
proteins  and peptides.

• First, the PRM uses the quadrupole  (Q1) to select the precursor ion,
and the selection window is usually m/z≤2; then, the precursor  ion is
fragmented in the collision cell (Q2); finally, Orbitrap replaces Q3,
 scans all product ions with high resolution and high accuracy.

• Generates high resolution, full scan MS/MS data. All transitions can
be used to confirm peptide ID. Don’t have to choose ions beforehand.

• It is most suitable for quantification of multiple proteins in  complex

sample.
 Basic workflow for analysis
Extract proteins Digest into peptides Chromatographic separation of peptides (C18 column)

ESI

Electrospray Ionization MS analysis

Q1 q2 Q3

Ion Intensity
Parent ion Fragmentatin Fragment
selection ion
selection Time
 General procedure to establish a targeted proteomic
experiment
1. Selection of target protein.
2. The selection of those peptide that present good MS response,
optimal signal intensity and uniquely identify the targeted protein.
3. Selection of predominant transitions to be used.
4. Optimization of specific MS parameters (e.g. the collision energy) to
maximize the signal response.
5. Validation of the assay to confirm peptide identity.
6. Quantification of targeted protein.
Transition selection and optimization is specifically associated with
SRM/MRM.
 Selection of target protein

• In targeted experiment, the researcher should have prior

knowledge of protein and their peptide behaviors which includes
the retention time and fragmentation pattern.

• This information is either present in literature, proteomics

repositories data base available online or have to be determined
before experiment through bottom up shotgun LC-MS/MS
experiment.

• Several, sites like Uniprot, Protein Atlas and Protein–protein

interactions IntAct are available freely to select the target
proteins.
 Selection of Target Peptides
• Proteotypic peptides can be selected from discovery-based
experiments or searching publicly available databases,
such as PeptideAtlas.
• PeptideAtlas is a useful resource for planning targeted
proteomics experiments.
• The data in PeptideAtlas contains information such as;
• Types of peptides
• Number of times they have been detected
• Fragmentation pattern in MS/MS spectrum
• Relative abundance of each fragment ions
• Computational tools can also be used for predicting
peptides.
 Criteria for Selection of Target Peptides
• Peptides should be unique to the protein of interest.
• Peptides should be easily detectable by LC-MS.
• Peptide length about 8-25 amino acids.
• Both ends of the peptide should be tryptic.
• Avoid missed cleavage sites.
• Avoid two enzymatic sites sequential to each other.
• Avoid frequently modified amino acids.
• Check for post translational modifications.
 In silico Prediction of Targeted Peptides

• Skyline is a software tool which performs in silico

digestion of target protein to predict either tryptic or non-
tryptic peptides depending on selected protease and
further filters peptides which fall within user specified
criteria defined by user.

• In Skyline, instrument specific methods can be easily

designed and exported for the instruments. Raw data
acquired with these instruments can then be imported
directly into Skyline for analysis.
 Selection of Transitions
• The combination of m/z setting for the first and third quadrupole is
referred to as transition.

• During selection one selects the fragment ions for each precursor-
ion charge state that provide the highest signal intensity and
lowest level of interfering signals to ensure effective quantification.

• Fragment-ion spectra from shotgun experiments have been used

to predict intense transitions.

• Such selection can also be guided by interfaces such as SRMAtlas

that access proteomic databases.

• There is commercially available software Skyline which generates

peptide transition in-silico.
 SRMAtlas
• A web-based query form of SRMAtlas accepts a protein accession
number as input to select a requested number of proteotypic
peptides and up to 10 respective SRM transitions in an instant.

• Resulting transition list is downloadable either as acquisition method

for a QqQ instrument or as a transition table importable to Skyline.

• Directly generated acquisition methods comprise experimentally

optimized CEs and default QqQ instrument settings.

• SRMAtlas enables the fastest transition from a protein target list to an

applicable SRM assay.
 Optimization for each transition to maximize signal
response/sensitivity

• Collision energy optimizing for each transition may increase overall

sensitivity however, is only really needed if sensitivity is very low or
below the limit of detection (LOD).

• After optimization of transitions, Validation of the MRM assay to

confirm peptide identity, e.g. by acquiring a full MS2 spectrum of the
peptide in the triple quadrupole instrument used for MRM.
 Validation of transitions by Standards
• Traditionally peptide validation was done with absolute
quantification of a single peptide by spiking a stable isotope labeled
(SIL) internal standard peptide (e.g., 13C, 15N) into samples of
interest.
• Then similarity between retention time, co-elution and
fragmentation characteristics between the two peptides (target vs.
standard) are evaluated. At the time of peptide elution, such
transition yield a perfect set of co-eluting intensity peaks.
• In the case of large experiments, where 10s to 100s of peptides are
monitored, we typically use a combination of spectral libraries ,
retention time prediction and manual inspection of elution profiles
for peptide validation.

• Skyline provides spectral libraries and correlation between

predicted and observed retention time for each peptide, making it
possible to review 100 peptides in minutes.
A method that uses crude synthetic peptide libraries and their
batch measurement on a QQQ-type instrument for SRM
development. This allows extraction and validation of the optimal
SRM transitions for ~100 peptides per hour with high confidence.
 Quantification by Targeted Method
• Quantification in targeted method (SRM or PRM) can be label-based
or label-free.

• In label-based, endogenous peptides are quantified by comparing

signals with the internal standards that are spiked into the samples
during processing.

• Heavy isotope-labeled synthetic peptides have the same

physicochemical properties as their endogenous counterparts. They
can be distinguished in a mass spectrometer based on the masses.

• Optimal amounts of internal standards are determined

experimentally by spiking different concentrations of the heavy-
labeled peptides into the samples.
• Intensity of peptide peaks depends on their physical properties,
absolute quantification is achieved by utilizing internal standard with
the same sequence at a known concentration.

• Apart from the difference in mass, all other chemical and physical
properties of the two peptides are identical; hence retention times in
LC, ionization efficiencies and fragmentation patterns in MS/MS are
all identical. Thus, quantitative determination of peptides of interest
can be carried out only by comparing the intensities of the two peaks.

• The peak area of each of the transitions of a precursor ion is

integrated and used for quantification.
 Proteins as Internal Standards

• To overcome the problem of synthesizing internal standard for each

peptide, heavy-labeled full-length proteins such as QconCAT are
used as internal standards.
• QconCAT is an artificial protein designed as a linear concatenation
of tryptic peptides. It collectively provides target peptides for
different proteins of interest.
• After enzymatic digestion, heavy-labeled peptides are released
from QconCAT. The peptides are identical to peptides released from
the digestion of corresponding endogenous protein.
Metabolic synthesis through concatemerization. Firstly, the proteotypic
quantification peptides that correspond to the target proteins are
selected and synthesized together as a single protein through the
corresponding synthetic designed gene. Gene expression can be
performed in a medium that contains isotopically enriched lysine and
arginine to produce isotope-labelled standards.
 Label-Free Targeted Method
• Label-free is a simple, straightforward and cost-effective method of
targeted quantification.
• An equal amount of samples are analyzed to estimate the relative
abundance of proteins across samples.

• Protein quantitation is performed using either ion peak intensity or

spectral counting.

• Relative quantitation by ion peak intensity relies on LC-MS only. The

direct MS m/z values for all ions are detected and their signal
intensities at a particular time recorded.

• Label-free relative quantitation by spectral counts entails comparing

the sum of the MS/MS spectra from a given peptide across multiple
samples, which has been shown to directly correlate with protein
abundance. 
LABEL FREE LABEL BASED
 Applications of Targeted Proteomics

• Cellular signalling pathways

• Biomarker discovery/Validation
• Differences in protein abundance due to genetic
variations
• Post-translational modifications
• Comparative analysis of proteins across different samples
under specific given conditions
 Representative applications of targeted
proteomics in biological and clinical studies.
Representative applications of targeted proteomics in
biological and clinical studies.
Preclinical verification of candidate protein
biomarkers by targeted proteomics assays
in 2012-2015.
Preclinical verification of candidate protein biomarkers by targeted
proteomics assays in 2012-2016.
Candidate protein biomarker Disease type Specimen Assay type
Candidate protein biomarker Disease type Specimen Assay type
Key studies covering a wide range of
applications of SRM-MS
 SRM/MRM
• SRM/MRM can achieve rapid, sensitive, specific quantification of
the target protein in a complex system and excellent quantitative
reproducibility.
• SRM/MRM technology eliminates most of the non-target
detection, that making the noise signal greatly reduced.
• Improves the detection sensitivity significantly.
• SRM requires significant effort in building a data acquisition
method.
• Selecting the best possible transitions for the target proteins
results in reliable quantification.
 PRM
• The mass accuracy can eliminate the background interference.
• Improve the detection limit and sensitivity  in complex background
effectively.
• Full scan of product ions, without the  need to select the ion pair
and optimize the fragmentation energy.
• Easier to  establish the assay.
• Elimination of effort required to develop and optimize the traditional
SRM assay.
• PRM spectra would be highly specific, all potential product ions are
available to confirm the identity of the peptide.
Basic characteristics of SRM and PRM in tandem mass
spectrometry acquisition methods
 Conclusion
• Targeted studies facilitate hypothesis-driven proteomic experiments
by exclusively focusing on quantitative monitoring of predefined sets
of proteins across multiple samples.
• Parallel reaction monitoring (PRM) and selected reaction monitoring
(SRM) are two main methods of choice for quantifying and validating
proteins across hundreds to thousands of samples.
• This analytical based method will surely be helpful in near future in
improving the human health by identifying those biomarkers which
are present in trace amount.
• Both high-sensitivity detection of low abundant proteins and
their quantification using this technique employ stable isotope
labeled peptide internal standards.
• SRM still has limitations in selectivity and requires more assay
development/validation for achieving high quality quantification
assays because it is operated on a low-resolution QqQ MS.
• PRM is a novel, targeted quantification method performed in a
high resolution and high mass accuracy mode on a quadrupole
Orbitrap mass spectrometer.
• It can be expected that its application will further expand and, in
the future, may supplant the low-resolution targeted method.
Thank You