Medical Genomics – Lecture 11 – Proteomics
Proteomics; Collection of proteins expressed in human cells
throughout life and under all conditions
Goal; identify and ascribe (benoem) function to proteins under all
biologically plausible conditions
The proteome is highly complex and many chemical modifications
are possible
Properties of a protein;
• Size; total number of amino acids
• Charge; average of basic and acidic amino acids
• Hydrophobicity; orientation of non-polar amino acids
Proteomics technologies;
• Protein separation on the basis of charge and molecular weight
o Polyacrylamide Gel Electrophoresis (PAGE); separate proteins within a
gel (- upside to + downside, proteins are
negative charged want to +). It is a network of
cross-linked molecules (acrylamide) that
forms a solid support where proteins can
migrate through
Proteins treated with SDS before electrophoresis SDS-
PAGE. SDS molecules bind to protein --> proteins lose
normal shape --> proteins become strongly negatively
shared. To make proteins move in an electric field based
on their mass difference
▪ 1D gel electrophoresis; separation on molecular
weight
▪ 2D gel electrophoresis; first separation by charge
(a change in the pH until it encounters a pH level
where its charge is neutralized), second separation
by molecular weight
o 2D liquid chromatography; first separation by charged,
second separation by hydrophobicity
The higher peak the more protein
• Protein detection and identification on the basis of their mass
o Mass spectrometry; how to identify proteins?
▪ Preparation of protein sample;
-Extraction from a gel OR liquid chromatography
-Digestion by proteases
▪ Mass spectrometry measures mass-
charge ratio of peptide fragments.
Components of mass spectrometer; ion
source, mass analyzer, ion detector, data
acquisition unit.
▪ Identified peptides are compared with
database
, First step is ionization (because mass spectrometer only able to
analyze ions, cleaving molecules to generate fragments with a ionic
charge)
Sources for ionization
1. ESI (electrospray ionization); used in conjunction with protein
separation techniques
2. MALDI (matrix-assisted laser desorption/ionization); does not
require separation of the protein sample and extracts ions form
proteins on the surface
Methods of analyzing;
1. Ion trap; used with ESI. Electric and magnetic fields
are used
2. Time of flight; used with MALDI. Ions with high mass-
to-charge ratio travel with lower velocity, the arrival
time is used to generate mass spectrum
Stable-isotope protein labeling technique to quantify differences in
protein expression
They are non-radioactive isotopes.
Because different isotopes of an element have different masses they
can have measurable effect on mass-to-charge ratio of an ionic peptide.
This leads to shifts in peak position
Example; isolate coded affinity tag (ICAT); label protein of interest with
ICAT --> isolate ICAT containing peptides --> liquid chromatography
purification --> mass spectrometry --> database search for protein
identification
Proteomics; Collection of proteins expressed in human cells
throughout life and under all conditions
Goal; identify and ascribe (benoem) function to proteins under all
biologically plausible conditions
The proteome is highly complex and many chemical modifications
are possible
Properties of a protein;
• Size; total number of amino acids
• Charge; average of basic and acidic amino acids
• Hydrophobicity; orientation of non-polar amino acids
Proteomics technologies;
• Protein separation on the basis of charge and molecular weight
o Polyacrylamide Gel Electrophoresis (PAGE); separate proteins within a
gel (- upside to + downside, proteins are
negative charged want to +). It is a network of
cross-linked molecules (acrylamide) that
forms a solid support where proteins can
migrate through
Proteins treated with SDS before electrophoresis SDS-
PAGE. SDS molecules bind to protein --> proteins lose
normal shape --> proteins become strongly negatively
shared. To make proteins move in an electric field based
on their mass difference
▪ 1D gel electrophoresis; separation on molecular
weight
▪ 2D gel electrophoresis; first separation by charge
(a change in the pH until it encounters a pH level
where its charge is neutralized), second separation
by molecular weight
o 2D liquid chromatography; first separation by charged,
second separation by hydrophobicity
The higher peak the more protein
• Protein detection and identification on the basis of their mass
o Mass spectrometry; how to identify proteins?
▪ Preparation of protein sample;
-Extraction from a gel OR liquid chromatography
-Digestion by proteases
▪ Mass spectrometry measures mass-
charge ratio of peptide fragments.
Components of mass spectrometer; ion
source, mass analyzer, ion detector, data
acquisition unit.
▪ Identified peptides are compared with
database
, First step is ionization (because mass spectrometer only able to
analyze ions, cleaving molecules to generate fragments with a ionic
charge)
Sources for ionization
1. ESI (electrospray ionization); used in conjunction with protein
separation techniques
2. MALDI (matrix-assisted laser desorption/ionization); does not
require separation of the protein sample and extracts ions form
proteins on the surface
Methods of analyzing;
1. Ion trap; used with ESI. Electric and magnetic fields
are used
2. Time of flight; used with MALDI. Ions with high mass-
to-charge ratio travel with lower velocity, the arrival
time is used to generate mass spectrum
Stable-isotope protein labeling technique to quantify differences in
protein expression
They are non-radioactive isotopes.
Because different isotopes of an element have different masses they
can have measurable effect on mass-to-charge ratio of an ionic peptide.
This leads to shifts in peak position
Example; isolate coded affinity tag (ICAT); label protein of interest with
ICAT --> isolate ICAT containing peptides --> liquid chromatography
purification --> mass spectrometry --> database search for protein
identification
