1Department of Chemistry, Tunghai University, 2Department of Biochemistry, University of California, Riverside
Hsu, Y., Traugh, J. A. Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation. J. Vis. Exp. (57), e3602, doi:10.3791/3602 (2011).
Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called γ-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.
1. Pak2 Activation
2. Identification of Pepsin-Digested Pak2 Fragments
3. Measurement of Amide H/D Exchange
4. Representative Results:
The caspase cleavage and autophosphorylation are verified by SDS-PAGE Commassie staining. Inactive Pak2 is a single band of 58 kDa. Caspase cleavage of Pak2 is complete, and produces two fragments, p27 and p34 which migrate separately. The migration of autophosphorylated p27 and p34 shows an overlap on the gel due to the retarded migration of the fully phosphorylated p27 fragment (Figure 2).
H/D exchange experiments are carried out multiple times between 0 and 10 min (Figure 3). As an example, the time course of deuterium incorporation into the m/z peak 1697.85 of inactive Pak2 is composed of multiple isotopic peaks as shown by the shift of the mass envelope over time.
Figure 1. Equation for the calculation of deuteration.
Figure 2. Autophosphorylation and caspase cleavage of Pak2. Caspase-cleaved Pak2 (left lane), intact inactive Pak2 (middle lane) and caspase-cleaved autophosphorylated Pak2 (right lane) were analyzed by SDS-PAGE and Coommassie blue staining. Adapted from Hsu, YH et al2.
Figure 3. Mass spectrum of a time course of deuterium incorporation for inactive Pak2. Inactive Pak2 was subjected to H/D exchange for 0-10 min and analyzed by MALDI-TOF. An example of the expanded isotopic distribution of MALDI-TOF of peak m/z 1697.85 during the time course of H/D exchange is shown. The red dotted line indicates the average of the mass envelope and the shift of the mass envelope shows the deuteration of a peptide.
Identification of the Pepsin-Digested Fragments is a critical step of the H/D exchange experiment. Incorrect identification of the peptide can lead to a wrong conclusion. Pepsin-digested Pak2 is eluted through a reverse phase HPLC C18 column to obtain a clean background. In our experiments, a total of 40 fractions were collected and subjected to MALDI-TOF. Multiple peptides could be identified in each of the fractions. The peptides were subjected to tandem mass spectrometry (Q-TOF MS/MS and oMALDI MS/MS) to identify their sequences. The MS/MS spectra of a peptide should have at least three product ions to confirm the identification of the peptide.
The Data Explorer 4.0 computer program (Applied Biosystems) performs the initial baseline correction and noise filtration. Each spectrum must be calibrated based on two sequenced peaks. We calibrate our spectrum by the theoretical mass of the undeuterated m/z which are 923.45 and 1697.84. The mass accuracy after calibration can reach 10 ppm. Upon deuteration, the mono-isotope may shift to a higher mass. Unitary step increases to these m/z ratios yielded the enhanced masses associated with deuteration. The peak intensities of the mass envelope will not affect the deuteration level. However, the peak intensities of the isotopic peaks in a particular mass envelope are critical for the calculation of the average mass. The first step of the calculation of the incorporated deuteron is subtracting the peptide mass of the non-deuterated sample from the deuterated sample. Three other factors, the D2O dilution, the residual deuterons in the side chains and the back exchange, will need to be further considered in the calculation (Figure 1). The D2O dilution is the dilution factor of D2O after mixing the sample and D2O buffer to initiate the H/D exchange. The residual deuteron (4.5%) in the side chains of the pepsin-digested peptides needs to be subtracted. The back-exchange is the inevitable loss of deuteration in all deuterated and pepsin-digested peptides in the mass measurement process.
The most solvent accessible peptide (m/z = 1105.60) after 24-hour of H/D exchange represents a full deuteration region. The incorporated deuteron number for each of the peptides is the difference between the centroid of the deuterated and non-deuterated Pak2 peptic peptides. The most highly deuterated peptide m/z 1105 was selected to represent full deuteration when Pak2 was at 24 hr of H/D exchange. The Back Exchange Ratio was calculated by the actual incorporated deuteron number at 24 hr of H/D exchange divided by all possible exchangeable sites at peptide m/z 1105.
No conflicts of interest declared.
This work is supported in part from the National Institutes of Health Grant GM-26738 (to J.A.T.).
|MALDI-TOF||PE Biosystems||Voyager DE STR|
|Q-TOF mass spectrometer||Q-TOF Ultima-Global|
|QSTAR XL oMALDI MS/MS||Applied Biosystems|
|Data Explorer 4.0 computer program||Applied Biosystems|