M. Cecilia Della Valle
Graduate Program in Molecular Biosciences
3rd Laboratory Rotation – January 26th to April 2nd, 2004
PI: Dr. Gaetano Montelione- CABM


H/D EXCHANGE MONITORED BY MASS SPECTROMETRY”


H/D exchange experiments monitored by NMR are used as a parameter of protein stability. This methodology, although very informative, requires high-tech equipment, a big amount of protein sample and a high level of expertise for data processing and evaluation.

The objective of the rotation project was to be able to monitor H/D exchange experiments by MALDI-TOF Mass Spectrometry (MALDI-MS). MALDI-MS requires less expensive equipment and a very small amount of sample. It is also less complex to operate and analyze the data, therefore being accessible to operators of different training levels. Last but not least, it gives the possibility of automation with robotics. This would make possible to analyze protein folding by stability measurements on a high number of samples in a short period of time, enabling the rapid identification of promising new proteins.

Using the paper of S. Ghaemmaghami et al (1) as starting point I started the optimization of the technique using Maltose Binding Protein (MBP) and the NH2 terminus of Lambda repressor (Lambda) purified by Tatiana B., as protein samples.

The first step was to determine the MALDI-MS system conditions and working concentrations for internal standards, Lambda and MBP. Afterwards, we decided and wrote an H/D exchange protocol using the conditions in the paper with some minor changes. I did the H/D exchange experiments with Lambda and MBP: some change in the m/z of the proteins in time was observed but the internal standard (BSA) showed high dispersion.

A main issue in preparing the sample for analysis of H/D exchange by Mass Spectrometry is to reduce the back exchange of the already exchanged Hydrogen, as much as possible. The pH of the matrix solvent, the temperature and the time of sample preparation are key points. Therefore, I tried to find the conditions to control each one of these parameters in order to reduce this effect.

After trying with internal calibration with no success I decided to work with external calibration using a double standard that lies within the m/z of the sample protein. The external calibration with double standard worked very well and reduced the SD of the data making it possible to observe the H/D exchange and the effect of other parameters (pH, temperature) in the system without interferences.

The pH of the standard matrix solvent (TFA 0.1%/ACN 50 %) is about 1.8. It is desirable that the pH of the aqueous component of the solvent is 3; that is the pH at which the lowest

H/D exchange rate is observed. We tried different buffers: Sodium Phosphate 20 mM, Sodium Acetate 25 mM, and Citrate/Succinate 25 mM. None of those seemed to work
well: it was very hard to get good signal from these samples, probably due to very low ionization of the samples. I decided to go on using the standard matrix solvent (TFA 0.1%/ACN 50 %) which gave better data.

In order to control the temperature all the reagents were kept in the cold. The matrix was aliquoted and kept on ice until the addition of the sample. The MALDI plate was kept at -20ºC ON and stabilized on ice for spotting. The addition of the sample and spotting were done very fast and under the hood to minimize the handling time.

The optimization of the procedure was done for MBP and then also applied to Lambda. The final Optimized protocol worked very well for both MBP and Lambda. The quality of the results improved substantially compared to the first experiment for both proteins: reproducible replicates and a consistent raise in the m/z with time was observed.
In the case of MBP less SD was observed in the data. In the case of Lambda, the SD was not a problem in the original data.

It is important to point out that the H/D exchange for Lambda was finally performed at pH 4.0 instead of pH 7.0. The first H/D exchange experiment performed at pH 7.0, showed a very fast rate of exchange and no difference in the delta m/z in time was observed. This was suggested by the NMR experiments, which showed a slower rate of exchange at pH 4.0 than at pH 7.0.

Outline of the experiments and tests done during the rotation
H/D Exchange Experiments monitored by MALDI-MS
1. MS-1 (30-1-04): determine work concentrations for standards, Lambda and MBP
2. MS-2/MS-3 (2/3/04):
Experiment #1: H/D exchange, MBP (pH 6.3) and Lambda (pH 7.0). Different time points, using internal standards: BSA as st. for MBP, and Insulin as a st. for Lambda.
a. A delta m/z of ~70 was observed in the non-calibrated MBP. BSA gave high SD, concern about calibrating with it.
b. A delta m/z of -40 was observed in the non calibrated Lambda. No change with time.
3. MS-4 (2-10-04): trying to optimize some parameters of the acquisition method.
4. MS-5 (2-11-04)
Experiment #2: MBP H/D exchange Different time points. Using matrix pH=3 with phosphate buffer (the pH measured in the final ACN/Buffer mixture)
a. A delta m/z of ~85 was observed in the non-calibrated MBP and a delta m/z of ~12 in the calibrated MBP. Still concern about BSA. Didn’t pay attention to the buffer
5. MS-6 (2-17-04)
Experiment #3: MBP H/D exchange with Guanidine. One time point: 60 minutes
a. A delta m/z of ~88 was observed in the non-calibrated MBP and a delta m/z of ~170 in the calibrated MBP. Lower signal and noisy data. Still concern about BSA.
Reading period.
6. MS-7 (3/3/04): Trying external calibration with Aldolase (~39 KDa) and Apomyoglobin (16 KDa).
a. Low signal but the data is less dispersed. External calibration works well.
7. MS-8 (3/5/04): Trying external calibration with Aldolase (~39 KDa) and Apomyoglobin (16 KDa)
a. External calibration works well.
8. MS-9 (3/12/04):
Trying buffers, Acetate and Citrate/Succinate
a. General low signal, even with TFA/ACN. Acetate seems to work very similar to TFA/ ACN with the sample. One of the Citrates gave no signal with MBP. Both buffers worked well with the standards
9. MS-10 (3-17-04)
Experiment #4: MBP H/D exchange experiment with External calibration using procedure for minimizing back exchange in the sample preparation for MALDI-MS. Compared Buffer acetate/ACN to TFA/ACN. Cold plate approach.
a. TFA/ACN worked very nice, good signal. The SD of the results was low. The replicates showed reproducibility of the data. A total delta m/z of ~144 Da was observed for MBP in 60 minutes, with a consistent raise in the m/z in time (no fluctuation). Conclusion: The external calibration with double standard worked very well and reduced the SD of the data. The crystallization of the matrix and the longer drying time under cold conditions were well tolerated: the “cold procedure” didn’t affect the quality of the data; in fact it made it seemed to have improved it.
b. Acetate buffer pH=3 in the matrix solvent didn’t work well: very low signal and fluctuating data. It’s not possible to analyze the effect of the pH in the back-exchange.

MS-11: Tatiana finished shooting the Acetate set b from MS-10
10. MS-12: disregard, H+ sample was not shot and we can’t compare
11. MS-13: (3-24-04):
Experiment #5: Tatiana did Lambda H/D exchange experiment at pH 4.0. using the “Optimized cold protocol”
a. TFA/ACN worked very nice, good signal. The SD of the results was low. The replicates showed reproducibility of the data, except for two data points which I assign to experimental errors. A total delta m/z of ~64 Da was observed for Lamda in 60 minutes, with a consistent raise in the m/z in time (no fluctuation). The “Optimized protocol” worked very well for Lambda too.



Mass Spec H-D Exchange Protocol II
Optimized Protocol: external calibration and cold target

Materials and reagents
Protein Samples
- Lamdba Repressor N-terminal domain (Lambda):
cc=13.92 mg/ml
MW~ 10 KDa
- Maltose Binding Protein (MBP):
cc= 24.72 mg/ml
MW= 40,826.4
External standards (MS Cal 3: Protein MALDI MS Calibration Kit)
Insulin, MS standard - SIGMA I-6279, lot (Avg M+H=5,734.51)
Aldolase MS standard - SIGMA A 9096 (Avg M+H= 39,212.28)
Apomyoglobin, MS standard - SIGMA A 8971 (Avg M+H= 16,952.27)
Albumin, Bovine, MS standard - SIGMA A-8471(Avg M+H=66,430.09)
The internal st stocks were reconstituted to100 pmol/ul with the appropriate solvent (see SIGMA kit insert)
Matrix: sinapinic acid (SA) from SIGMA
Exchange buffer: 20 mM sodium phosphate/20 mM sodium acetate/100 mM NaCl, pH 6.3 for MBP and pH 7.0 for λ.
MALDI Plate: pre cool the target ON at -20ºC. Place on ice on top of a plastic box and let stabilize for half an hour before spotting.
Solvents: Acetonitrile (ACN), Trifluoroacetic Acid (TFA), D2O, H2O
Procedures
Matrix solution preparation: weigh 10 mg of SA or use one of the tubes provided with the kit. Add 0.5 ml of ACN, 1 ul of TFA and 499 ul of H2O.
Standards: Prepare standards for external calibration.
- For MBP: make two dilutions of the standard
Standard I: Standard II
Aldolase st 1 ul Standard I 20 ul
Apomyoglobin st 1 ul SA in solvent 20 ul
SA in solvent 38 ul Final volume 40 ul
Final volume 40 ul

- For Lambda: make two dilutions of the standard
Standard I: Standard II
Insulin 1 ul Standard I 20 ul
Apomyoglobin st 1 ul SA in solvent 20 ul
SA in solvent 38 ul Final volume 40 ul
Final volume 40 ul
Keep everything on ice (2ºC) before the addition of the protein.
Exchange buffer: 20 mM sodium phosphate/20 mM sodium acetate/100 mM NaCl, pH 6.3 for MBP and pH 4.0 for Lambda
.
Prepare from 10 X stocks of buffer with D2O, adjust pH with DCl and NaOD if necessary.
Hydrogen exchange experiments:
1. To 4 ul of protein sample (use samples undiluted) add 96 ul of deuterated exchange buffer.
Temperature: 24 ºC in water bath
Time: 0, 10’, 20’, 30’, 40’, 50’, 60’
2. At every given time take 1 ul of each exchange reaction (take 2 aliquots for each time point). Add 1.0 ul of the exchange reaction to 19 ul of the ice cold matrix solution. Vortex briefly.
Immediately spot 1 ul of the sample in matrix on a MALDI plate and dry as fast as possible under the hood (under these cold conditions the samples take around 4-5 minutes to dry). Also spot 1 ul of the standard preparations.
3. Data collection - MALDI- TOF. Voyager 5.0:
a- Acquire the standard and generate a calibration file
b- Open the generated calibration file and acquire the samples: for each sample. Obtain 5 independent spectra, 100 shots each.
c- Process the data (*) in Data Explorer 4.0 and using the average of the five spectra plot a graph of m/z versus time.

*Data Processing: Under Peak Detection use: For m/z over 20 KDa use a mass resolution of 1,000. For m/z less than 20 KDa use a mass resolution of 2,000.

Processing steps: 1- Baseline correction
2- Noise Removal (2SD)
3- Noise Filter smooth: Default
4- Centroiding
Notes:
All the data are in a file called Rotation Project under NSEG on RONG PRIME 1-014 in the network.
Some people have reported working with Citrate/Succinate buffer pH 2.5 (3). I would suggest trying a couple of more times or buffers in order to get a better control of the back exchange.
Drying time of the sample: I would suggest finding a way to apply an air current to the spotted samples to lower the drying time.

References
1- A quantitative high-troughput screen for protein stability” S. Ghaemmaghami et al. PNAS. July 18, 2000. Vol 97. No 15
2- “Delayed Extraction Time-of-flight Mass MALDI Spectrometry of Proteins above 25000 Da”. U. Bahr, J. Stahl-Zeng, E. Gleitsmann and M. Karas* Journal of Mass Spectrometrt, Vol. 32, 1111-1116 (1997)
3- “Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back-exchange effect” Marc Kipping1. and Angelika Schierhorn2.
J. Mass Spectrom. 2003; 38: 271–276