Dit document omvat de info van de slides plus mijn lesnotities van les 5 van concepts, gegeven door Xaveer Van Ostade. De lessen van Kurt Boonen (Les 2,3,4 en 8) heb ik ook, maar de notities staan op de slides. Moest je hierin ook geïnteresseerd zijn, mag je me altijd contacteren via messenger :))
Lecture 10: 11/12
Applica'ons
Protein iden*fica*on
- Past: Purifica-on of protein -> digest -> Edman degrada-on: laborious!
- Top-down strategy (1):
o Determine MW of the protein (if resolu-on is high enough) -> sufficient when no
PTM present.
- BoHom-up strategy (2):
o Trypsinise protein and iden-fy on the basis of PMF and/or PFF (or de novo)
o ANer digest only a limited number of pep-des has PTM, hence iden-fica-on is
possible.
o Top down
- Protein not know -> de novo sequencing or sequencing parts of the protein and then
produce oligonucleo-des for further cloning
- Iden-fica-on without prior separa-on can be performed on less complex protein
mixtures (max. 5-6 proteins/sample).
o Mixture not too complex -> immediately introduce your sample in the mass spect
without prior isola-on or purifica-on and immediately perform iden-fica-on on
the complex
- Sample is more complex -> purifica-on by chromatography or gel electrophoresis
Detec*on and characteriza*on of muta*ons
- Muta-ons result in MW differences, varying between 0.0364 Da (Gln/lys) and 129.0578
(Gly/Trp)
- Three steps:
o (Protein MW determina-on (high resolu-on and accuracy necessary))
o Enzyma-c digest for determina-on of mutated pep-de (based upon MW
difference)
o Sequence determina-on of mutated pep-de
- One AA replaced by another AA -> MW of pep-de will differ -> you can look for
pep-des that differ according to the molecular weight of that pep-de in databases
- Example Separa-ng alfa and beta chains
o MALDI-TOF: detects muta-ons in the b-chain (mutant chain is 14 Da heavier as
compared to na-ve chain)
o ANer tryp-c digest of the β chains MALDI-TOF shows two new pep-des: T9m and
T8+T9m (results from missed cleavage).
m/z T9m = 1683.90 = 14.01 Da heavier as compared to normal pep-de T9 =
1669.89.
o Since the difference of 14.01 Da corresponds to 6 different muta-ons (G/A, S/T,
V/I, V/L, N/Q of D/E; see table) and since al these AAs (G, S, V, N en D) are present
in the pep-de T9 (AZ sequence 67-82), the nature of the muta-on cannot solely
be determined from the mass difference.
o MS/MS is necessary for sequence determina-on of T9m. Conclusion: Asp79
replaced by Glu
, o Some pep-des are ok and some of them are split up -> normal and mutant
present.
o Muta-on in B chain -> it looks like you have a slit of the B globin chain
o Pep-des; some are ok, and other are spliHed
§ T9; normal and mutant form are present
§ T8 + T9: trypsiniza-on didn’t occur
o Difference of 14.01 Da -> many of the mutant form are present in the pep-de T9
-> we s-ll don’t know which AA have been replaced -> sequence the T9 mutant
pep-de -> Asp79 replaced by Glu
Verifica*on of structure and purity of proteins and pep*des
- Series of peaks -> protein of 14.590 Da -> p18 (MW = 14.589 Da)
- However: two other series are present:
o Small T one (8%): MW = 12.651 Da. Corresponds to C-terminal part of p18 aNer
splicing at posi-on 111-11
o Small D one (3%): MW = 29.175 Da. Corresponds to dimer generated aNer
disulfide bridge forma-on between two p18 proteins.
Non-covalent protein complexes and 3D structure informa*on
Na#ve-denatured:
- Distribu-on of charges in denatured protein is broader + more charges are present.
This is the result of the exposi-on of larger protein surfaces allowing the ioniza-on of
plenty of basic AAs.
- Example: acidic denatura-on of human myoglobin in func-on of -me -> 3 species are
observed:
o na-ve hMb (haem + myoglobin)
o denatured hMb (haem + denatured myoglobin)
o denatured aMb (denatured apomyoglobin = without haem moiety)
- Simple method to find out something about the structure of the protein. When the
proteins is denatured -> protein is unfolded and many more AA are acceptable for
protona-on -> will have more charges as compared to the unfolded proteins
- You know something about the folding of the protein compared to the complete folded
form.
Accessibility
- Accessibility of a certain AA for the solvent can be inves-gated by reac-on of the
protein with reagents that cause irreversible reac-ons, specific for the AA side chain.
Number and posi-on of the modified AAs determines posi-on in the protein 3D
structure.
o When the has AA has covalently bonded groups the MW will increase -> will be at
the surface of the proteins
Internal sites
- Internal sites are protected by bulky groups and will be trypsinized less easy. Hence the
masses of the tryp-c pep-des will give informa-on about the loca-on of some K and
R residues.
o Treat sample, your purified protein with limited amount trypsin -> will cut at the
surface of the protein but there is not enough trypsine to further cut the protein,
only the outside of the protein will be trypsinized -> which pep-de will appear?
These lysins and arginines will be at the outside of the protein
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