What is proteomics?
- The global / large scale study of proteins
- Comparable to DNA for genomics
- Big difference: unlike DNA, amplification of proteins is not possible
- The term proteome was first used in 1997
- The proteome is the entire set of proteins produced or modified by an organism or system
- While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically used
for mass spectrometry applications with or without protein purification
- The field is full of acronyms!
- SILAC, iTRAQ, ESI, MALDI, TOF, TMT, SRM, MS1, MS2 etc
- Importantly it can be used to not just define the components of systems but to quantify them
The mass-spectrometry (MS) /proteomic experiment:
- An experiment comprises many steps coupled together. Sample preparation, enzymatic digestion,
chromatography, ionisation, MS, informatics (not just database matching)
- All steps are critical to the successful outcome of the experiment
- A variety of quantitative workflows are available
- No standardised method agreed
- A proteomics workflow does not stop when you have a list of peptides / proteins
- Informatic analysis required e.g. statistics for abundance / volcano plots / pathway analysis / principal component
analysis (PCA) / hierarchical clustering / protein-protein interaction networks
, The liquid-chromatography (LC) –tandem mass
spectrometry (MSMS) experiment
Liquid-Chromatography (LC) is a method that is
coupled inline to allow separation of peptides before
ionisation
- MALDI – Matrix Assisted Laser Desorption
Ionisation - Lower throughput
- ESI – ElectroSpray Ionisation - Higher throughput
- most popular method
- HPLC – High-Performance (High-Pressure) Liquid
Chromatography
MS measures mass to charge ratios (m/z) (of
peptides)
Peptide sequencing and database matching
- Peptides are selected in the MS for
fragmentation
- Peptides fragment at the peptide bond in a
characteristic way
- Terminology for ions is abc and xyz
- Fragmentation at various positions along the
peptide backbone generates fragments of
different lengths
- The peaks seen are a sum of many copies of the
selected molecule snapping in different places
- Matching MS spectra to a database is a
probability-based exercise
Approaches of quantitative proteomics:
Relative or absolute quantification?
- The global / large scale study of proteins
- Comparable to DNA for genomics
- Big difference: unlike DNA, amplification of proteins is not possible
- The term proteome was first used in 1997
- The proteome is the entire set of proteins produced or modified by an organism or system
- While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically used
for mass spectrometry applications with or without protein purification
- The field is full of acronyms!
- SILAC, iTRAQ, ESI, MALDI, TOF, TMT, SRM, MS1, MS2 etc
- Importantly it can be used to not just define the components of systems but to quantify them
The mass-spectrometry (MS) /proteomic experiment:
- An experiment comprises many steps coupled together. Sample preparation, enzymatic digestion,
chromatography, ionisation, MS, informatics (not just database matching)
- All steps are critical to the successful outcome of the experiment
- A variety of quantitative workflows are available
- No standardised method agreed
- A proteomics workflow does not stop when you have a list of peptides / proteins
- Informatic analysis required e.g. statistics for abundance / volcano plots / pathway analysis / principal component
analysis (PCA) / hierarchical clustering / protein-protein interaction networks
, The liquid-chromatography (LC) –tandem mass
spectrometry (MSMS) experiment
Liquid-Chromatography (LC) is a method that is
coupled inline to allow separation of peptides before
ionisation
- MALDI – Matrix Assisted Laser Desorption
Ionisation - Lower throughput
- ESI – ElectroSpray Ionisation - Higher throughput
- most popular method
- HPLC – High-Performance (High-Pressure) Liquid
Chromatography
MS measures mass to charge ratios (m/z) (of
peptides)
Peptide sequencing and database matching
- Peptides are selected in the MS for
fragmentation
- Peptides fragment at the peptide bond in a
characteristic way
- Terminology for ions is abc and xyz
- Fragmentation at various positions along the
peptide backbone generates fragments of
different lengths
- The peaks seen are a sum of many copies of the
selected molecule snapping in different places
- Matching MS spectra to a database is a
probability-based exercise
Approaches of quantitative proteomics:
Relative or absolute quantification?
