SFP5: Protein Characterisation And Primary Structure Determination Flashcards
Are native or denatured molecular weights larger?
Native are greater than denatured
Can subunits be determined with native and denatured mwts?
Yes
Name four methods of molecular weight determination?
- Need a ‘pure’ protein sample
1) gel filtration - native
2) SDS PAGE - denatured
3) analytical ultracentrifugation - native, very accurate
4) mass spectrometry- very accurate
How to determine native molecular weight determination?
Gel filtration
Run mixture of standard proteins of known native Mwt
Standard curve allows ‘unknown’ native Mwt to be estimated
How to determine number of subunits using native and denatured mwt
Number of subunits = native mwt/denatured mwt
Provided subunits are identical
What are the two methods for determining amino acid sequences?
1) Edman degradation
2) Mass spectrometry
How does peptide mass fingerprinting (PMF) work?
1) cut out protein spot of interest from 2D
2) proteolytically digest in gel with trypsin (or other enzyme) - cuts after K and R
3) peptide mixture, AAs have different sequences, AAs have absolute mass values
4) ionise and measure peptide masses by ms (protein identification)
Which sequencing method (Peptide mass fingerprinting or tandem MS) is used to when the gene sequence is already known?
Peptide mass fingerprinting
What does the first analyser in tandem MS generate?
A peptide fingerprint
What does the collision cell do in tandem MS?
Selects an ionised peptide, fragments it further
What does the second analyser do in tandem MS?
Analyses the fragmented peptide to generate AA sequence data
Can isoleucine and leucine be determined apart in MS/MS peptide sequencing
No
What two amino acids are the same in mass?
glutamine and lysine (but wont have k in sequence as it will cut just after the k)
