add the appropriate pre - tested amount of caspase 3 to 20 l of 12 mg / ml pak2 in buffer a ( 50 mm tris - hcl , 150 mm nacl , and 2 mm dtt at ph 7.5 ) and incubate at 34 c for 30 min to fully cleave and activate pak2 .
analyze the cleavage products by sds - page ( 10 % ) and coommassie blue staining .
add the cleaved pak2 to activation buffer b ( 50 mm tris - hcl , 150 mm nacl , 15 mm mgcl2 , 10 mm atp ) at ph 7.5 , in 20 l to a final concentration of 10 mg / ml pak2 , and incubate at 34 c for 30 min to fully activate pak2 by autophosphorylation at 8 sites .
confirm full cleavage of pak2 by migration of the p34 and p27 fragments as determined by sds - page .
proper verification of the autophosphorylation conditions is made via autoradiography in a separate experiment using ( p)atp or mass spectrometry to detect phosphopeptides .
add 1 ml of 0.1 % trifluroacetic acid ( tfa ) at ph 2.5 to wash the agarose beads twice prior to use .
then , centrifuge the samples for 15 sec at 2,000 x g to remove the beads .
add pak2 ( 20 g in 2 l of 150 mm nacl , 50 mm tris - hcl and 2 mm dtt at ph 7.5 ) to 100 l of 0.1 % tfa , and incubate the sample for 5 min with 30 l of pepsin - conjugated agarose beads .
equilibrate a reverse phase hplc c18 column with a primary solvent system of 0.1 % tfa at ph 2.5 .
the pepsin digest ( 50 l ) is eluted using a linear 100-min gradient of 0 - 80 % acetonitrile containing 0.1 % tfa at a flow rate of 0.2 ml / min .
dry the hplc fractions with a speed - vac ( 3 h ) and resuspend the samples in a solution of acetonitrile ( 5 l ) and 0.1 % tfa ( 5 l ) at ph 2.5 .
initially , each fraction is screened for peptides using the maldi - tof perseptive biosystems voyager de str ( pe biosystems , foster city , ca , usa ) . the 16 fractions containing peptides are subjected to tandem mass spectrometry using a q - tof mass spectrometer and a qstar xl omaldi ms / ms to identify the fragments .
the sequence of each peptide is verified by the product ions generated by the tandem mass spectrometry with the theoretical m / z ratios based on the primary sequence of pak2 using the protein prospector website ( http://prospector.ucsf.edu/ ) .
hold all solutions and reagents needed for h / d exchange at 25 c in a water bath .
atp is dissolved in water and the ph is adjusted to 7.0 prior to use .
the additional 4 mm atp in the d2o buffer is to prevent atp dissociated from pak2 after dilution .
initiate h / d exchange by addition of 18 l of buffered d2o ( 50 mm mops ph 6.98 , 125 mm nacl ) to 2 l ( 20 g ) of pak2 in a buffer containing 150 mm nacl , 50 mm tris - hcl ( ph 7.5 ) and 2 mm dtt , resulting in a pak2 level of 10 mg / ml and a ph of 7.0 at 0c .
incubate the samples in triplicate in the h / d exchange for 0 , 0.5 , 1 , 3 , 5 , and 10 min , or 24 h. quench the h / d exchange process by adding 180 l of ice - cold 0.1 % tfa at ph 2.2 , which brings the ph to 2.5 in a final volume of 200 l . for the samples containing atp , 180 l of ice - cold 0.11 % tfa at ph 2.2 is used to maintain the ph at 2.5 in the quenched condition . add an aliquot ( 100 l ) of each quenched sample to 30 l of activated pepsin - conjugated agarose beads .
the pepsin digestion is allowed to proceed on ice for five min and the samples are vortexed every 30 sec .
terminate the digestion by centrifugation of the sample for 15 sec at 5,000 x g at 4 c to remove the pepsin .
rapidly freeze the samples in liquid n2 and store at -80 c for no more than 2 days prior to maldi - tof analysis .
the matrix for maldi is 5 mg / ml of -cyano-4-hydroxycinnamic acid ( chca ) dissolved in a solution ( 1:1:1 ) of acetonitrile , ethanol and 0.1 % tfa at ph 2.5 and held at 0 c . partially thaw each sample quickly and mix 1 l of each sample with 1 l of matrix and spot on a 4 c maldi target plate .
apply a moderate vacuum to the plate to dry the spots in 30 sec prior to maldi - tof analysis .
acquire data at a 2-ghz sampling rate at 100,000 data channels , with a 20,000-v accelerating voltage , and a 65 % grid voltage using delayed extraction with a 180-ns pulse delay .
the maldi - tof instrument is not restricted to any brand or any particular software .
calculate the back exchange based on the ratio of the actual incorporated deuteron number at 24 hr of h / d exchange divided by all possible exchangeable sites , which are the backbone amides except the n - terminal amide ( figure 1 ) .
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 .
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 .
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 .
add the appropriate pre - tested amount of caspase 3 to 20 l of 12 mg / ml pak2 in buffer a ( 50 mm tris - hcl , 150 mm nacl , and 2 mm dtt at ph 7.5 ) and incubate at 34 c for 30 min to fully cleave and activate pak2 .
analyze the cleavage products by sds - page ( 10 % ) and coommassie blue staining .
add the cleaved pak2 to activation buffer b ( 50 mm tris - hcl , 150 mm nacl , 15 mm mgcl2 , 10 mm atp ) at ph 7.5 , in 20 l to a final concentration of 10 mg / ml pak2 , and incubate at 34 c for 30 min to fully activate pak2 by autophosphorylation at 8 sites .
confirm full cleavage of pak2 by migration of the p34 and p27 fragments as determined by sds - page .
proper verification of the autophosphorylation conditions is made via autoradiography in a separate experiment using ( p)atp or mass spectrometry to detect phosphopeptides .
add 1 ml of 0.1 % trifluroacetic acid ( tfa ) at ph 2.5 to wash the agarose beads twice prior to use . then , centrifuge the samples for 15 sec at 2,000 x g to remove the beads .
add pak2 ( 20 g in 2 l of 150 mm nacl , 50 mm tris - hcl and 2 mm dtt at ph 7.5 ) to 100 l of 0.1 % tfa , and incubate the sample for 5 min with 30 l of pepsin - conjugated agarose beads .
equilibrate a reverse phase hplc c18 column with a primary solvent system of 0.1 % tfa at ph 2.5 .
the pepsin digest ( 50 l ) is eluted using a linear 100-min gradient of 0 - 80 % acetonitrile containing 0.1 % tfa at a flow rate of 0.2 ml / min .
dry the hplc fractions with a speed - vac ( 3 h ) and resuspend the samples in a solution of acetonitrile ( 5 l ) and 0.1 % tfa ( 5 l ) at ph 2.5 .
initially , each fraction is screened for peptides using the maldi - tof perseptive biosystems voyager de str ( pe biosystems , foster city , ca , usa ) .
the 16 fractions containing peptides are subjected to tandem mass spectrometry using a q - tof mass spectrometer and a qstar xl omaldi ms / ms to identify the fragments .
the sequence of each peptide is verified by the product ions generated by the tandem mass spectrometry with the theoretical m / z ratios based on the primary sequence of pak2 using the protein prospector website ( http://prospector.ucsf.edu/ ) .
hold all solutions and reagents needed for h / d exchange at 25 c in a water bath .
atp is dissolved in water and the ph is adjusted to 7.0 prior to use .
the additional 4 mm atp in the d2o buffer is to prevent atp dissociated from pak2 after dilution .
initiate h / d exchange by addition of 18 l of buffered d2o ( 50 mm mops ph 6.98 , 125 mm nacl ) to 2 l ( 20 g ) of pak2 in a buffer containing 150 mm nacl , 50 mm tris - hcl ( ph 7.5 ) and 2 mm dtt , resulting in a pak2 level of 10 mg / ml and a ph of 7.0 at 0c .
incubate the samples in triplicate in the h / d exchange for 0 , 0.5 , 1 , 3 , 5 , and 10 min , or 24 h. quench the h / d exchange process by adding 180 l of ice - cold 0.1 % tfa at ph 2.2 , which brings the ph to 2.5 in a final volume of 200 l . for the samples containing atp ,
180 l of ice - cold 0.11 % tfa at ph 2.2 is used to maintain the ph at 2.5 in the quenched condition .
add an aliquot ( 100 l ) of each quenched sample to 30 l of activated pepsin - conjugated agarose beads .
the pepsin digestion is allowed to proceed on ice for five min and the samples are vortexed every 30 sec .
terminate the digestion by centrifugation of the sample for 15 sec at 5,000 x g at 4 c to remove the pepsin .
rapidly freeze the samples in liquid n2 and store at -80 c for no more than 2 days prior to maldi - tof analysis .
the matrix for maldi is 5 mg / ml of -cyano-4-hydroxycinnamic acid ( chca ) dissolved in a solution ( 1:1:1 ) of acetonitrile , ethanol and 0.1 % tfa at ph 2.5 and held at 0 c .
partially thaw each sample quickly and mix 1 l of each sample with 1 l of matrix and spot on a 4 c maldi target plate .
apply a moderate vacuum to the plate to dry the spots in 30 sec prior to maldi - tof analysis .
acquire data at a 2-ghz sampling rate at 100,000 data channels , with a 20,000-v accelerating voltage , and a 65 % grid voltage using delayed extraction with a 180-ns pulse delay .
the maldi - tof instrument is not restricted to any brand or any particular software .
calculate the back exchange based on the ratio of the actual incorporated deuteron number at 24 hr of h / d exchange divided by all possible exchangeable sites , which are the backbone amides except the n - terminal amide ( figure 1 ) .
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 .
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 .
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 .
pepsin - digested pak2 is eluted through a reverse phase hplc c18 column to obtain a clean background . in our experiments ,
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 .
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 .
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 .
 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 -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 . 
Protocol
1. Pak2 Activation
2. Identification of Pepsin-Digested Pak2 Fragments
3. Measurement of Amide H/D Exchange
4. Representative Results:
Discussion
Disclosures