1. Lecture 9 Web: pollev.com/ucibio Text: To: 37607
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2. Lecture 9 objectives Understand principles of protein sequencing
Understand principles and applications of MS
Understand assumptions and limitations of MM kinetics
Understand experimental determination of MM parameters

3. Protein structure and isolation
Column chromatography principles
Gel electrophoresis ( non-denaturing and SDS)
Proteomics

4. Differences in the “proteome”

5. Sequencing proteins

6. Sequencing proteins

7. Sequencing proteins is a step-wise process

8. Sequencing proteins

9. Protein sequencing Why cut up protein prior to sequencing?
Why multiple ways of cutting up protein?

10. Protein sequencing NB-B-C-A E-R-X-A B-A-Q-N S-A-F-EC NB-B-C-A-
C-A-B-A
C-A-B-A
Q-N-E-R-X
A-S-A-F-EC
B-A-Q-N
Q-N-E-R-X
E-R-X-A
A-S-A-F-EC
S-A-F-EC

11. Sequencing proteins

12. Mass spectrometry (Mass spec)

13. MS Example

14. MS-MS to sequence proteins

15. Random fragmentation A-B-C-D A-B-C-D A-B-C-D A-B-C-D A- B-C-D A-B- C-D
Signal intensity
m/z
A-B-C
A-B-C-
A-B- A
A-B-C-D
A-B-C-D

16. MS Example

17. Using MS to generate a “proteome”
Copyright  2013 Pearson Canada Inc.

18. Using MS to generate a “proteome”
Copyright  2013 Pearson Canada Inc.

19. Protein purified, now what?
What do we want to know about our protein (Hexokinase)?

20. Simplifying kinetics Initial rate = [P] ≈ 0 Practically 100% ES  P
At GIVEN [E], rate ∝ on [S]

21. Michaelis-Menten kinetics: A model
Makes several assumptions:
1. _________________ (Vo)
2. At given [__]
3. [__] ≫ [__]
4. At steady state:
ES formation=ES breakdown

22. Determining Km V 0 = V Max [S] S + K m S + K m = V Max [S] V 0
S + K m = V Max [S] 0.5 V Max
S + K m = 2[S]
K m = [S] when V o = ½ V Max

23. The Michaelis-Menten kinetics graph

24. Measuring Initial Velocity at different [S]

25. The Michaelis-Menten kinetics graph

26. How to estimate VMax?
y = mx + c

27. The Lineweaver-Burk plot

28. The turnover number (k2 or kcat)
Turnover number = number of S molecules  P per second

29. Turnover number Eg: k2 = 1000 molecules sec-1
1/k2 = 1/1000 sec molecule-1
1/k2 = sec molecule-1