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Introduction to Proteomics, NGS, LC-MS - Coggle Diagram
Introduction to Proteomics
Human Proteome Atlas
Aim to catalogue all estimated 37trillion cells that make up human body
Better disease management, therapeutics
The proteome
Definition
Profiling the entire set of proteins within a system/cell at a given time and under defined conditions
Dynamic, differs over time
Reflection of the transcriptome (expression of genes)
Enables an accurate image of cellular state
Proteins consist of amino acid sub units
Leu and Ile have same chemical formula but diff. structure
Even at high resolution, mass will be exactly the same
MS1 scan will be the same
MS2 scan (fragmentation) will be different
Omic hierarchy
Genome
Transcriptome
Proteome
Metabolome
Phenotype/net result
Mechanism
Organisation + control
Everything that could happen
Exist in complement to each other
Feedback mechanisms
4 fundamental omics levels
Proteins
Size
kDa = 1000Da = 1000m/z
Big
Lots of active sites
Changeable
On different scale to metabolites
Mass analysers
Orbitrap system
Combination of mass spec architectures
TOF
Scan and fragment at same time
Very high mass resolution
Time of Flight
Triple Quadrupole
Quadrupole
Simplest
Not typically used in proteomics (mass range low)
Quadrupole TOF
better mass filtering + higher resolution
Ion trap
Quadrupolar field
Applied voltage pf field related to mass
All used to precisely manipulate ion and characterise it
Scan speed
How fast the analyser can scan the entire range of mass spectrum
Higher resolution + lower speed
Mass resoltuion
Ability to resolve closely related adjunct mass peak
Bigger number + higher resolution
MS aquisiton
Parallel/multiple reaction monitoring
Single ion monitoring
Set mass spectrometer to very specific mass
Look at a few proteins e.g. 5
Discovery or validation
Discovery is proteomics
Use data to produce hypothesis
DDA/DIA
Structural info embedded into scan
Look at seevrl hundred prpteins at once
Limitations of MS based proteomics
Technologies only sample a fraction of all proteins present in a sample
Only a fraction of all ID'd proteins can also be reliably quantified
For every 1000 proteins, use around 300-400 proteins in analysis
Bottom-up proteomics approach
1) Extraction of protein content from biolpgicsl samples
2) Subsequent determintation of approximate protein
3) Fractionation (SDS gels) + trypsin digestionq
4) Clean up and lyophilisation
5) Chemical analysis
6) Database matching + peak table sequencing
7) Data analysis + interpretation
Remove trypsin + stop reaction
Trypsin has ability to cleave proteins precisely + reputably
Instrumentation
LC
Electrospray ionisation
MS
Convert liquid stage to gaseous stage
Apply charge to end of spray needle
Chatge is absorbed by solvetns and induce coloumbic explosion event
Solvent evaporates a lot quicker than analyte
Flow rate is very low (300nL/min)
Solvent consumption is very low (mls/week)
Fragmentation nomenclature
B ions extend from N-terminus
Y ions extend from C-terminus
Advantages
Disadvantages
Top-down proteomics
In tact protein analysis
Sample must be ultra pure
Too many proteins confuse system
MALDI
Sandwhich substrate attenuates
Advantaes
Look at intcat proteins
Disadvantages
Sample prep
Trypsin digestion
Cleave proteins into peptides
Gel based methods
SDS-PAGE
Load proteins into gel
Separated by Mw
Each protein band does not represent one protein but a class of prpteins at that weight
Allows sample to be profiled
Cheap
Protein estimation
Different assays used
Bradford
Produce calibration curve
Differential gel electrophoresis
Post-translational modification
Design of experiments
Methodology
SILAC
NGS
LC-MS