Lecutre 9 - Proteomic analysis Flashcards
What are the disadvantages and advantages of comparative SDS Page analysis?
Advantage:
Visual comparison
Easily presented and protein purified during analysis
Disadvantages:
Ineffective for low abundant proteins
Reproducibility?
Time consuming
What is 2D fluorescence difference gel electrophoresis?
2D Gel electrophoresis 3 different samples: Sample 1 Sample 2 Internal standard
3 Different stains (different absorbance wavelength) for each sample
Spot comparison:
Data quantitation
Image analysis (overlay images)
What are mass labels? (HPLC)
Differential proteomics
Introduction of different mass labels in the proteome
Mix the samples
Digest entire proteome
For each peptide, the mass differs due to labels
Comparitive height in Gel-electrophoresis reflect their ratio.
E.g.
Norma (1) and Fat (2) mouse protein analysis
Protein extraction from blood, label 1 with 12C and 2 with 13C
Mix
Trypsin digestion
LC/MSMS
Heavy and light of the SAME proteins will co-elute
Look at relative abundance
Upregulated peptides found in 2? (vice versa)
What is Isotope coded affinity tag (ICAT)?
HPLC
An isotopic labelling method used for quantitative proteomics by MS.
It uses chemical labelling agents containing 3 elements:
a reactive group for labelling AA side chain
a isotopically coded linker
a tag (biotin)
For quantitative analysis, one sample is labelled with the isotopically light probe (d0) and the other with isotopically heavy version (d8)
Both samples are combined and digested to minimize error
What is Stable isotope labelling by amino acids in cell culture (SILAC)?
SILAC utilizes heavy and light amino acids to label samples.
Each sample can have different modifications via. heat shocking
e.g
Sample 1 grown normally
Sample 2 heat shocked and modified.
Mix samples
Digest
MS analysis and comparison
What are the advantages of ICAT and SILAC?
Advantages:
automated
Gives accurate relative abundance
Detect low abundant proteins
Disadvantages:
Produce complicated spectra (x2 peaks)
Proteins must contain specific amino acids
Expensive
What is Isobaric tags for relative and absolute quantification (iTRAQ)?
(MSMS)
iTRAQ is a tag for peptides iTRAQ consists of 3 groups: Reporter group Balance group Peptide reactive group
The tagging of different peptides results in all of them being isobaric. The reporter groups have different masses and these are compared
What are the advantages and disadvantages of iTRAQ?
Advantages:
Automated
Gives relative abundance accurately
Detect low abundance proteins
Disadvantages:
Expensive
Need suppliers specific MS to do it
How are 13C labelled peptide internal standards made?
Convert peptide sequences into oligo DNA sequences
Splice together the individual oligo DNA’s to form a composite cDNA
Insert cDNA into a plasmid and recombinantly express in bacteria in the presence of Lys-13C,15N
Treat with trypsin
Regained orginal peptides made with 13C
What is MALDI imaging?
By using a spray coat instead of a droplet.
It looks at the distribution of peptides/proteins
It can classify different tissues
Increased leaser resolution allows more detail to be observed
Under what conditions direct purification is optimal?
When it is easy to purify complexes
e.g. ribonucleoproteins (RNA and proteins) purified by sucrose gradient ultrafiltration due to their size.
Under what conditions Poly/mono clonal antibodies purification is optimal?
Have antibody to one of more areas of the protein
Under what conditions Antibodies to epitope purification is optimal?
If no antibody is available, genetically add something that has one i.e an antibody recognised polypeptide.
Done by fusion of N or C term in a plasmid construct
Under what conditions affinity purification to fusion tag (GST) purification is optimal?
Glutathione-S-transferase (GST) fusion tags
Protein of interest is fused to GST (genetic level) and expressed.
GST section easily to immobilise on beads/column
Pour cell lysate through column
proteins that interact will stick
Eluted after washing by salt
Under what conditions tandem affinity purification is optimal?
Non specific binding to antibodies to uncomplexed proteins.
TAP-TAGGING
Have 2 affinity tags fused to proteins
Forms a complex
Purify by antibody to tag 1
Re-purify protein with antibody to tag 2
