Download Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name: Mass

Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name:_____________________________
Mass Spectrometry (Dr. Weintraub)
Use the Domon and Aebersold paper listed below as your primary resource. The Marx Nature
Methods “News Feature” may be useful to help you answer the questions. Note that in the
Domon and Aebersold paper, the instrument used for many of the analyses is a triple quadrupole
Higher performance instruments (in terms of mass resolution and mass accuracy) are often now
used for the same purpose; however, the underlying experimental design is the same. While you
are certainly welcome to include information about specific instrument configurations in your
answers, it is not necessary to do so. Please answer in your own words. Do not copy text
directly from a paper or other resource. Use citations when appropriate.
Domon, B. and Aebersold, R. Options and considerations when selecting a quantitative
proteomics strategy. Nat. Biotechnol. 2010 Jul;28(7):710-21. doi: 10.1038/nbt.1661. Epub 2010
Jul 9.
Marx, V. Targeted Proteomics. Nat Methods. 2013 Jan;10(1):19-22.
1. Briefly describe the analytical strategy used for (a) discovery, (b) directed, and (c) targeted
proteomics.
2. For each of the three techniques, briefly outline an experiment that would use that technique.
Include a discussion of the type of data that would result.
3. What is the role of stable isotope labeled internal standards? How are they selected? When
are they added to the samples? What different types are available?
4. How does targeted proteomics differ from SILAC in terms of protein identification and
quantification?
Point breakdown:
1. 30 points
2. 30 points
3. 20 points
4. 20 points
Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name:_____________________________
Hydrodynamics (Dr. Demeler)
The shape of a linear, double-stranded DNA molecule without any bound water can be
approximated with a long rod model with a diameter of 2 nm. The rise per base pair is 0.34 nm,
assume an average molar mass for a base pair of 660 g/mol. In aqueous solution, the molecule
has a partial specific volume of 0.55 ml/g. Assume a solvent density of 1 g/ml and a viscosity of
0.01 p, and a temperature of 20°C, The gas constant is 8.314x107 erg/(K*mol), and Avogadro's
number is 6.0221x1023. The frictional ratio for a molecule matching a long rod model can be
estimated based on this relationship: f/f0 = [(2/3)1/3 P 2/3]/[ln(2P) – 0.30], where P is the axial ratio,
and P = a/b, and a is the half-length of the rod and b is the radius of the rod. Calculate the
following metrics for a 50, 100, 150, 200, 250 bp DNA molecule:
1. the length, l
2. the axial ratio P
3. the frictional ratio, f/f0
4. the sedimentation coefficient, s
5. the frictional coefficient, f
6. the diffusion coefficient, D
7. the Stokes radius, rs
8. the minimal sphere radius, r0
9. the minimal frictional coefficient f0
10. Check to see if the numbers change as you would expect as a sanity check for your program.
Indicate if there is an increase, decrease, or no change with increasing number of basepairs for
f/f0, s, D, vbar, anhydrous density, and frictional coefficient. (10 points)
Report your answers in the form of a short C++ program that I can compile on bcf.uthscsa.edu
(let me know if you have trouble logging in). The program should report the results in narrative
format like this:
The length of the DNA molecule is ….
The frictional ratio of the DNA molecule is … (etc…, order doesn't matter)
Tips:
* There are no trick questions, but read the problem carefully, and watch the units! Make sure the
numbers make sense and have the right order of magnitude. Use cm.gram.second (cgs) units.
* for calculations treat integers as floating point variables. Example: write: 2.0/3.0 instead of 2/3
* The constant π is available in the include file “math.h” under the name “M_PI” (e.g.,
area=M_PI*r*r).
* REMEMBER: C++ is case SENSITIVE!
* To exponentiate, you can use the pow() function like this:
float val = pow (float num, 1.0/3.0); // this takes the cube root of the number “num” and
places the result into the variable “val”.
I have copied a skeleton program to each user account called “report.cpp”. Please modify this
program to add the additional logic needed to solve these problems.
Each correct calculation is worth 10 points.
Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name:_____________________________
Protein Interactions (Dr. Lafer)
You have discovered a novel protein involved in clathrin mediated endocytosis which u name
endobender. You find that endobender interacts with the phosphoinositide PI(4,5)P 2.
Endobender is a monomer of 60,000 Dalton, while PI(4,5)P2 is 1042 Dalton.
1. Design a kinetic SPR experiment to quantify the on and off rates of the interaction between
endobender and PI(4,5)P2. What will be the analyte and what will be the ligand? How will you
decide how much ligand to immobilize, what flow rate to use, and how long to inject the
analyte?
Once you determine the association and dissociation rate constants, k a and kd, explain how you
would use this information to calculate the equilibrium dissociation constant KD?
2. Design an equilibrium SPR experiment to directly measure the equilibrium dissociation
constant KD? What will be the analyte and what will be the ligand? How will you decide how
much ligand to immobilize, what flow rate to use, and how long to inject the analyte?
How will the design of this experiment be different from the kinetic experiment in part 1?
3. You also decide to characterize the interaction by performing an ITC experiment. Would you
expect the KDs you get from the three experiments to be the same or different? If they are the
same what would you conclude? If they are different what would you conclude and which one is
most likely to be correct and why? What other quantities will you be able to calculate from the
ITC experiment that you were not able to calculate from the SPR experiments?
Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name:_____________________________
Hydrodynamic modeling (Dr. Brookes)
a. [15 points] Where does
?
b. [20 points] What information is required in a bead model that will be used to compute
hydrodyamic parameters?
c. [20 points] Summarize advantages and disadvantages of the three methods discussed for
computing hydrodynamic parameters.
Biophysics Exam #2 – due Monday, 5/16 at 5 pm. Name:_____________________________
Small angle scattering (Dr. Brookes)
a. [20 points] In a SAXS experimental setup, suppose the original maximum q value recorded on
the detector is 1 Angstrom-1 and we would like data out to a q value of 2 Angstrom -1 . Describe
two different ways to modify the experimental setup to achieve this?
b. [25 points] Suppose you have a rigid 2 domain protein connected by a linker. The domains
are approximately a cylinder and a sphere. Draw the expected plot of the P(r) derived from a
SAS experiment. Be sure to label the “x” axis appropriately.
100 Angstroms