proteomic profiling based on enzyme activity is assuming significance in drug discovery as it becomes possible to profile selectivity of drugs and their mechanism of action .
such an approach focuses on protein function , an aspect which has been missing from expression proteomics . a functional proteomic profiling approach has the potential not only to help identify targets for diagnosis and therapy , specifically in personal medicine [ 3 , 4 ] , but also to reveal the underlying mechanisms of action of disease - sustaining proteins .
methods for global analysis of protein expression and function , including liquid chromatography with mass spectrometry ( ms ) for shotgun analysis [ 6 , 7 ] , yeast two - hybrid methods , and protein microarrays , have been crucial in developing the field of proteomics , but they do not provide an accurate assessment of functional states of proteins in cells and tissues .
activity - based protein profiling ( abpp ) was first demonstrated for serine hydrolyses   and has now been applied to other enzyme classes such as kinases , phosphatases , and histone deacetylates [ 10 , 12 ] .
abpp typically uses active site - directed covalent probes to interrogate specific subsets ( families ) of enzymes in complex proteomes to provide a quantitative assessment of the functional state of individual enzymes in the family .
the probe - bound enzymes can be visualized with sds - page or purified using affinity tools for peptide or labeling site identification with ms .
although this approach is promising , it is limited by the availability of suitable synthetic probes . also , while abpp categorizes the active site in enzymes , it does not measure the functional kinetics of enzymes and therefore can be considered only as an indirect measure of protein function .  
it forms the central component of the workflow strategy , which has the potential to identify functional biomarkers from natural cellular sources .
the proposed method would fill an unmet need for research in drug response and biomarker discovery for investigations in natural cellular source environments .
the physiological relevance of working with natural cellular sources is especially significant for discovery , which targets proteins whose function may be altered by post - translational modification , noncovalent regulatory factors or splice variants . such an approach may help to reconcile data from high - throughput screening of recombinant proteins to natural cellular sources .
it is anticipated that select subproteomes will be subjected to downstream characterization by liquid chromatography mass spectrometry ( lc - ms ) , and other suitable identification methods in common use , so as to annotate sequence and structure to function .   while the term functional proteomicsencompasses a variety of phenotypic descriptions of known or measurablefunctional consequencesincluding cellular response to stimuli   and binding interactions [ 14 , 15 ] , etc . , the model approach reported herein is limited to characterizing enzyme kinetic properties .
the proposed profiling strategy starts with subfractionation of complex proteomes using serafile   ( uspto 20040106131 , profact proteomics , monmouth junctions , nj , usa ) .
this proprietary protein separations platform is configured as a surface library with associated interrogation methods designed to retain bioactivity of the samples . as a result
, subproteome pools obtained after serafile separations can be characterized for their enzyme activity properties ( e.g. , enzyme activity with and without inhibitors , activators , or cosubstrates ) .
then , a collective functional profile of the original proteome is generated as an integrated profile of the functional properties of the characterized subproteomes .
this approach provides multiple data points to characterize and compare samples , thereby increasing the robustness and reliability of analysis .
this profiling method can compare one proteome sample to another ( intersample analysis , e , g . , tissue type versus tissue type , or normal versus diseased tissue ) and can compare different subproteomes of the same complex proteome ( intrasample analysis ) .  
the cyclic nucleotide phosphodiesterases ( pdes ) enzyme family has been used in this study as a model class of proteins to demonstrate the proposed strategy .
pdes are enzymes that hydrolyze the second messenger adenosine 3 , 5-cyclic monophosphate ( camp ) or guanosine 3 , 5-cyclic monophosphate ( cgmp ) , or both .
these small molecules along with other nucleotides , lipids , and ions function as secondary messengers .
the second messenger camp mediates a wide variety of actions of hormones and neurotransmitters and influences cell growth , differentiation , survival , and inflammatory processes .
class i pdes ( found in protozoa and metazoa ) are camp specific ( pde4 , 7 and 8) or cgmp specific ( pde5 , 6 and 9 ) or can hydrolyze both camp and cgmp ( pde1 , 2 , 3 , 10 , and 11 ) [ 17 , 19 , 20 ] . a comprehensive review of pdes can be found in [ 17 , 19 , 21 ] .  
pdes are widely acknowledged and explored as drug targets in pulmonary , neurodegenerative , and vascular diseases , and in diabetes , osteoporosis , cancer , rheumatoid arthritis , and depression .
inhibitors of pde5 and pde3 are already in clinical use , but numerous other pde inhibitors have not been used for therapeutic purposes due to side effects such as nausea and emesis . the proposed approach to proteomic profiling
is guided by the principle that , by discriminating and characterizing pde variants in natural sources , greater disease - specific therapeutic inhibition / activation can be achieved along with a better understanding of disease pathway dynamics .
this research article demonstrates functional proteomic profiling of camp - hydrolyzing phosphodiesterases from bovine and rat brains .
although earlier studies have documented the presence of different types of pdes in rat and bovine brains , a comprehensive comparative profile of pde proteomes based on function and content / identity has not been established .
it is known that bovine brain exhibits calmodulin - activated pde activity ( pde1 ) , as well as pde2 , and pde4 activity [ 25 , 26 ] .
the camp hydrolysis activity of pdes in bovine brain can be stimulated   or inhibited   by cgmp .
studies on rat brain have identified calmodulin - stimulated pdes [ 2931 ] ( pde1 ) , as well as pde4 isoforms [ 3234 ] .  
serafile was first applied for fractionation of each brain homogenate ( sample proteome ) into subproteomes , in order to reduce the complexity of pdes in the sample proteome .
then , these subproteomes were interrogated for camp hydrolysis activity in the presence and absence of cgmp .
cgmp is another substrate of pdes and is used as a challenge condition in these experiments .
the results were compiled into a signature profile of camp hydrolysis characteristics of each sample proteome , defined as an integrated profile of characteristics of serafile - generated subproteomes .
the hypothesis was that serafile and associated interrogation methods would generate distinct profiles of enzyme catalyzed camp hydrolysis - activities from bovine brain and rat brain homogenates because these are different mammalian species .
  the serafile inventions   encompass the surface characteristics and protocols suitable for differential proteomic fractionation .
each surface architecture was designed to have moderate binding capacity and was prepared with nugel epoxy ( biotech support group inc . , monmouth junction , nj , usa ) .
the epoxy - coated silica was modified by reacting it with different ligands to generate unique surfaces selectivities and was based on the premise that important ligand protein interactions include hydrogen bonds , ionic interactions ( salt bridges ) , hydrophobic interactions and ring structures .  
table 1 illustrates the differences in the properties of the surfaces in the library ; however for proprietary protection , details of the surface chemistries remain undisclosed .
an initial screen of 13 surfaces from the library ( table 1 ) and one underivatized control was performed .
further study was limited to a set of five surfaces ( a , b , d , m , and n ) from the surface library because the subproteomes obtained from these surfaces had the most distinguishing characteristics ( enzyme activity and its response to rolipram / vinpocetine / calmodulin , protein concentrations , and sds profile , data not shown ) .  
rat brain homogenate ( rbh ) and bovine brain homogenate ( bbh ) were supplied by lampire biologicals ( pipersville , pa , usa ) .
whole bovine or rat brain was homogenized in a prechilled blender using 100  ml of extraction buffer for every 50  g of brain tissue .
extraction buffer for bbh was 0.1  m tris , 2  mm edta , and ph 7.5 , and for rbh it was 1  mm edta , 10  mm hepes , and ph 7.4 .
each extraction buffer was made with protease inhibitor cocktail ( roche , indianapolis , in , u.s.a . ) .
homogenized brain - buffer mixtures were centrifuged at 4c , and the supernatant was used for the experiments .  
rbh and bbh samples were mixed with cleanascite ( biotech support group , monmouth junction , nj , u.s.a . ) in a 1  :  16 ratio of cleanascite - to - homogenate , to remove lipids and particulates .
clarified homogenates were obtained by following mixing and centrifugation steps as given in the manufacturer 's protocol .   
the pretreated homogenates were each subjected to separation by five serafile surfaces ( a , b , d , m , and n ) [ 3538 ] . for separation of each homogenate ,
50  mg of each surface contained in a spin - x tube ( corning inc .
, corning , ny , u.s.a . ) was equilibrated with binding buffer ( 0.05  m hepes , 1  mm mgcl2 , and ph 6.5 ) .
clarified bbh and rbh were diluted in the binding buffer , to ph 6.5 - 6.6 , and 200 l of each diluted homogenate ( load , 1.16  mg of total protein ) was added separately to each of the five surfaces , mixed for 10  mins , and then centrifuged .
( note that the total protein amounts used for serafile separations were based on the sensitivity of the camp hydrolysis assay used in our experiments for downstream analysis .
the serafile methodology is nevertheless amenable to protocols that can use g amounts of protein loads ) .
the flowthrough was collected as the 1st serafile fraction , represented as subproteomes a1 , b1 , d1 , m1 , and n1 , from surfaces a , b , d , m , and n , respectively .
the proteins bound on the surfaces were eluted with 200  l of elution buffer ( 0.05  m hepes , 1  mm mgcl2 , 0.5  m nacl , and ph 8.0 ) using mixing and centrifugation steps as above .
the flow - through collected in this process was the 2nd serafile fraction , represented as subproteomes a2 , b2 , d2 , m2 , and n2 , from surfaces a , b , d , m , and n , respectively .
mixing steps were performed using a mixmate ( eppendorf , hauppauge , ny , u.s.a . ) at 1150  rpm following an initial pulse of mixing on a vortex mixer .
centrifugation steps were performed using a tabletop centrifuge at 16873  rcf for 3  mins .
each brain homogenate , bovine and rat , was used for separations in triplicates .   
activity of camp hydrolysis in each subproteome was measured using a real - time kinetic assay [ 39 , 40 ]
. this assay links camp hydrolysis to nadh oxidation using coupling enzymes ( adenylate kinase , pyruvate kinase , and lactate dehydrogenase ) , and nadh loss can be measured at 340  nm . for each assay ,
a mixture of reaction buffer and coupling enzymes was equilibrated at room temperature for 16  mins ( stage i ) .
then , subproteomes were each individually added to the reaction mix , and loss in absorbance was measured for 16  mins ( stage ii ) . finally , substrate camp or a mix of camp and cgmp
was added to the assay , and the loss in absorbance was measured as above ( stage iii ) .
final concentrations of assay components were as follows : 9  mm mgcl2 , 0.46  mm cacl2 , 46  mm kcl , 46  mm hepes , 1  mm phosphoenolpyruvate , 46  m atp , 0.4  m nadh , 50  m camp , 0.8 units pyruvate kinase , 4 units lactate dehydrogenase , and 0.06 units adenylate kinase ( sigma , st .
louis , mo , u.s.a . ) with 6.25  l of each enzyme sample .
the camp hydrolysis activity of each sample was measured as the basal activity ( in the absence of cgmp ) and as challenged activity ( in presence of 25  m cgmp or 50  m cgmp ) .
volume - normalized enzyme assays were performed on each replicate subproteomes in a 96-well format using a multiskan mmc346 plate reader ( thermo scientific , hudson , nh , u.s.a . ) .   to measure camp hydrolysis activity in the unfractionated brain homogenates ,
the clarified homogenates were diluted with binding buffer to obtain 5 - 6 dilutions of each homogenate , which were then used for the assays as described above .  
protein content of all rbh and bbh proteomes and subproteomes was measured using a bca assay kit ( pierce , rockford , il , u.s.a . ) .
( a ) the camp hydrolysis activity of each sample was calculated as follows : 
 ( 1)enzyme  activity  ( nmoles  ml1 
min1 )  = path  length  correction  factorcorrected  pde  rate  ( min1)reaction  volume  ( ml)dilution  factormolar  absorption  coeffcient  ( m1  cm1)sample  volume  ( ml ) , 
 
 where corrected pde rate is a340 
nm(min ) stage ii , molar absorption coefficient is 1.25  10  m  cm , dilution factor is 1 , and path length correction ( to 10  mm ) is 3.16 .
( b ) the % change in camp hydrolysis activity of each sample was calculated as follows :  
 ( 2)%  change  in  camp  hydrolysis  activity=(challenged  enzyme  activitybasal  enzyme  activity)100basal  enzyme  activity , 
 
 where challenged enzyme activity is camp hydrolysis activity in presence of cgmp , and basal enzyme activity is camp hydrolysis activity in absence of cgmp .
  cleanascite [ 4144 ] , a solid - phase , nonionic adsorbent for lipid removal , significantly improved the clarity of brain homogenates and eliminated clogging of the surfaces during the serafile process . a 1  :  16 ratio of cleanascite to untreated bbh gave optimal results , with minimum loss of camp hydrolysis activity .
consequently , the same ratio of cleanascite to brain homogenate was used for rbh clarification .  
enzyme activity and protein analysis of the unfractionated brain homogenates showed that the mean specific activity of clarified rbh and bbh was comparable , between 8 and 8.8  units / mg , measured at 50  m camp concentration .  
a comparison of the basal and challenged camp hydrolysis activities in the dilutions of each homogenate is shown in figure 1 .
as expected , increase in activity ( basal and cgmp challenged ) was observed with increasing concentration of clarified homogenates .
the comparison also shows that at relatively lower concentrations of the homogenates , cgmp inhibited camp hydrolysis , while , at higher concentrations , camp hydrolysis activity increased .
in addition , the change in camp hydrolysis activity was more pronounced in the presence of 50  m cgmp than 25  m cgmp .
specifically , at 50  m cgmp ( figures 2(a ) and 2(b ) ) , the change from inhibition to activation of camp hydrolysis activity occurred above ~1.5  mg / ml protein in rbh and above 4  mg / ml protein in bbh proteomes .
thus , it is a characteristic in the pdes of rbh and bbh , that the effect of cgmp on camp hydrolysis is a function both of the concentration of the homogenate and of the concentration of cgmp .  
buffer - diluted , protein - normalized , and clarified rbh and bbh samples were used for separations .
each subproteome obtained was analyzed for protein content and camp hydrolysis activity under basal and cgmp - challenged conditions , and then the change in camp hydrolysis activity was calculated . to ensure that the observed properties of the subproteomes were not an effect of the dilution of the sample proteome ,
the properties of rbh and bbh proteomes and their respective serafile subproteomes were compared ( figures 2(a ) and 2(b ) ) .
the data in figure 2(a ) show that not all rbh subproteomes follow the activity versus protein content relationship of the rbh proteome .
data show that some subproteomes do share the activity versus protein content relationship of the sample proteome , likely indicating a comparable distribution of camp hydrolyzing pdes to total proteins .   to generate an intersample functional proteomic profile of rbh and bbh proteomes , the change in camp hydrolysis activity of each subproteome due to cgmp challenge was calculated and plotted as shown in figure 3 .
a functional proteomic profile of the brain homogenates in these experiments is defined by the collective response of individual serafile subproteomes to cgmp challenge .
a comparison between the functional profiles of the two homogenates ( figure 3 ) shows that , overall , these two profiles have a similar pattern ( i.e. , % change in camp hydrolysis is similar , positive or negative , in comparable fractions of the two homogenates ) .
however , a major difference is found in subproteome m2 of rbh and bbh ( refer to figure 3(a ) versus figure 3(e ) , and figure 3(c ) versus figure 3(g ) . subproteome m2 of rbh shows over 190% increase in camp hydrolysis in the presence of cgmp ( both 25  m and 50  m ) , while subproteome m2 of bbh shows over 90% decrease in camp hydrolysis in the presence of cgmp ( both 25  m and 50  m ) .
thus , subproteome m2 is a differentiating feature of this inter - sample analysis and therefore can be used for further sample characterization .   
these model data demonstrate that our proposed methods of protein separation generate subproteomes that are sufficiently differentiated for intersample functional analysis . as a result
, these methods can be potentially applied to effectively differentiate functional properties of complex proteomes and can be used to localize subset of proteins attributable to sample - specific responses .
the localized proteins can then be used for further analysis , characterization , and subsequent ms identification ( gene sequence annotation / reconciliation ) .
serafile separations use mild - to - moderate elution conditions with buffers like phosphate or hepes that are commonly used in the laboratory .
in addition , the solid - phase surface ( 50   derivatized silica ) can be easily removed by filtration .
thus serafile separations methods do not introduce substances like urea or sds that may restrict downstream compatibility with existing reporting assay and lc - ms detection methodologies .
therefore , the proposed methodology is considered to have a broad scope of applicability within the pathway to identification and can be potentially applied to profile narrowly defined therapeutically important classes of enzymes such as kinases or cyclic nucleotide phosphodiesterases .  
another important characteristic of serafile separations methodology is its reproducibility at different protein loads . in separate experiments , surface separation of 0.25  mg to 1  mg protein per 50  mg of surface
the reproducibility in sample separation can be significant for heterogeneous samples of clinical origin .  
incremental increase in ph was applied for enrichment of alkaline phosphatase ( data not shown , ) with an enrichment factor up to 20x .
the applications of serafile separations can be based on two basic types of sample and data analysis of ( i ) intersample analysis whereby samples such as tissues , cellular models , or biofluids are compared and contrasted and ( ii ) intrasample analyses , or differential analysis within a sample whereby the subproteomes are monitored with respect to a challenging modulation condition such as in drug response profiling .
inter - sample analysis of complex proteomes , as demonstrated , potentially applies to disease and nondisease comparisons , to identify differences in samples by compartmentalizing the most distinctive subproteomes associated with disease .
deeper characterization of these fractions with enrichment ( e.g. , with ph optimization of serafile separations , or with conventional separations 2de or hplc ) , followed by lc - ms analyses , can help identify prospective biomarkers .
it is important to recognize that any biomarker panel selected in this context would require more characterization , with larger sample sets and statistical validation .
intra - sample analysis , on the other hand , can be used to catalogue or index the effects of functional modulation of the daughter subproteomes .
this will be especially valuable for establishing localized panels of proteins that are responsive to modulation with drug compounds , with the same caveats as the aforementioned inter - sample analyses .  
the two data analysis strategies , profiling between samples , and cataloging within samples , are complementary insofar as molecular profiles that characterize and compartmentalize drug - responsive proteins from complex mixtures , can potentially , through coincident iterations with disease profiling , create a bulls - eye effect for drug repurposing .  
we envision that , for the drug development industry , the proposed methods for localizing proteins with known functional attributes offer new resources for biomarker discovery , complementing conventional methods of identification and sequence annotation . for
drug compounds , a challenge / response method , as described , can help address the problems of drug promiscuity and discern the subtleties of protein attributes ; when the same or similar underlying sequences , have multiple conformations and functions , and when different sequences sometimes perform the same or similar function .   as a way to begin sifting through these biological complexities , a more efficient method to characterize protein function and
starting with the enrichment of prospective functional biomarkers in localized subproteomes , we suggest that structural and sequence relationships can be determined .
such an approach has the potential to provide new and useful service to biomarker discovery and personalized medicine .
 functional proteomic profiling can help identify targets for disease diagnosis and therapy . 
 available methods are limited by the inability to profile many functional properties measured by enzymes kinetics . 
 the functional proteomic profiling approach proposed here seeks to overcome such limitations . 
 it begins with surface - based proteome separations of tissue / cell - line extracts , using serafile , a proprietary protein separations platform . 
 enzyme kinetic properties of resulting subproteomes are then characterized , and the data integrated into proteomic profiles . as a model , serafile - derived subproteomes of cyclic nucleotide - hydrolyzing phosphodiesterases ( pdes ) from bovine brain homogenate ( bbh ) and rat brain homogenate ( rbh ) 
 were characterized for camp hydrolysis activity in the presence ( challenge condition ) and absence of cgmp . 
 functional profiles of rbh and bbh were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes . 
 intersample analysis showed that comparable profiles differed in only a few data points , and that distinctive subproteomes can be generated from comparable tissue samples from different animals . 
 these results demonstrate that the proposed methods provide a means to simplify intersample differences , and to localize proteins attributable to sample - specific responses 
 . it can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling . 
1. Introduction
2. Materials and Methods
3. Results and Discussion