differential ion mobility 
 spectrometry ( dims ) provides a gas - phase 
 separation of ions prior to mass analysis . dims has the ability to 
 separate isobaric and isomeric species and therefore can improve the 
 specificity and detection limits of mass spectrometry - based analyses . low electric field ion mobility separations take advantage of the 
 difference in the collisional cross - section of ions , which is directly 
 proportional to the low - field ion mobility . at 
 low electric fields however , at high electric fields ( > 10 v / cm ) , the mobility 
 of an ion becomes dependent on the electric field in a complex function 
 that is still not understood . dims takes advantage 
 of the change in ion mobility in high electric fields to separate 
 ions based on the difference in ion mobility between low and high 
 electric fields . the gas flow into the mass spectrometer carries ions through 
 the gap between the dims electrodes . in transparent  
 mode , both of the electrodes are held at the same potential , and all 
 ions are allowed to pass through the device without an ion mobility 
 separation . in  active  mode , an asymmetric waveform 
 is applied to the electrodes . the dims waveform alternates between 
 low and high electric fields of opposing polarity , such that the ions 
 zigzag through the device . the v0-p of the dims waveform measured during the high electric field portion 
 is called the dispersion voltage ( dv ) . because ions have a different 
 mobility in the high field versus low field , they are displaced toward 
 one or the other electrodes during transit through the device . to 
 direct ions of a selected differential ion mobility into the mass 
 spectrometer , a compensation voltage ( cv ) must be applied to one of 
 the electrodes , which counter balances the displacement due to the 
 differential mobility . because high - field ion mobility is not yet 
 well understood , the optimum cv and dv for a given analyte are not 
 easily predicted . this makes dims currently most useful for targeted 
 analyses , where optimal dims conditions for the selection of the analyte 
 of interest can be determined prior to analysis of the real sample . 
 while there is a loss in overall ion signal using dims , the background 
 signal ( chemical noise ) is typically reduced to a much greater extent 
 than the analyte signal because background ions are eliminated through 
 collisions with the dims electrodes while target ions are not . the 
 net result is an overall increase in signal - to - noise ( s / n ) . these antigens consist of a 910 amino acid peptide 
 fragment , derived from a cancer associated protein , which is complexed 
 to a class i human leukocyte antigen ( hla ) molecule . the peptide / hla 
 complexes are assembled in the endoplasmic reticulum and subsequently 
 transported to and presented on the cancer cell s surface . 
 a cytotoxic cd8 t - cell expressing a t - cell receptor with 
 high binding affinity for the specific peptide / hla complex can kill 
 the antigen - presenting cells ( i.e. , cancer cell ) through release of 
 cytotoxic granule proteins . because of the high activity and 
 specificity of cancer - antigen specific cd8 + t - cells and their ability 
 to persist in patients indefinitely , many forms of cancer immunotherapy 
 have been developed to harness this approach in various cancers including 
 melanoma , renal cell carcinoma , prostate cancer , and leukemia . the identification of appropriate peptide antigens is a vital part 
 of the development of new t - cell therapies because of the complexity 
 and costs involved with the production and development of these therapeutics 
 and the risks involved with directing t - cells to noncancer ( i.e. , 
 normal tissue ) targets . the majority 
 of cancer antigenic peptides have been confirmed using 
 nontargeted reverse phase high - performance liquid chromatography coupled 
 to tandem mass spectrometry ( hplc  ms ) approaches on a complex 
 mixture of antigenic peptides obtained from a cellular sample . while this approach has proven extremely useful for peptide discovery , 
 nontargeted hplc ms preferentially identifies the most prevalent 
 peptides , which are not necessarily the most immunogenic . in recent years computational algorithms have 
 been developed to predict which peptides in a protein are likely to 
 bind specific hla molecules . these algorithms , when combined with the significant advances in 
 genomics and computational biology , can be used to predict the relatively 
 small number of antigenic peptides in tumor extracts that are likely 
 immunogenic and cancer - specific because they are produced as a result 
 of cancer - specific processes such as somatic gene mutations , fusions , 
 overexpression , and alternative splicing . by employing a computationally predictive cancer - antigen discovery 
 approach , targeted ms methodologies aimed at detecting cancer - specific 
 peptide antigens would facilitate the discovery of high - value cancer 
 peptide antigens . in this study , ms / ms was used to identify known cancer - associated 
 antigenic peptides from a human leukemia cell line without using hplc . a panel of 95  flashpure  
 peptides ( new england peptide , gardner ma ) that had been computationally 
 predicted to bind with high affinity to hla - a*0201 ( supplementary table s-1 ) were dissolved in 100% dmso to a 
 concentration of 20 mm and subsequently diluted to 1 m using 
 50/49.9/0.1 acn / h2o / fa ( v / v / v ) ( sigma - aldrich , st . louis , 
 mo ) ( supplementary table s-1 ) . to create 
 a peptide pool , 94 of the 20 mm peptide solutions in dmso were combined 
 in equal volumes and diluted to a final concentration of 1 m 
 of each peptide in 50/49.9/0.1 acn / h2o / fa ( v / v / v ) . the 
 95th peptide , glr ( amino acid sequence flssanehl ) , was further diluted 
 using 50/49.9/0.1 acn / h2o / fa ( v / v / v ) and spiked into the 
 94 peptide pool to make 1 m , 100 nm , 10 nm , 100 pm , 10 pm , 
 and 1 pm concentrations of glr peptide solutions . the u937 
 leukemia cell line stably transfected with hla - a*0201 ( called u937.a2 ) 
 were grown under sterile conditions at 37 c with 5% co2 in a medium consisting of rpmi 1640 supplemented with 10% heat inactivated 
 ( 56 c for 30 min ) fetal bovine serum ( fbs ) , 1% penicillin four liters 
 of u937.a2 cell culture suspension was incubated until the live cell 
 density reached 7.5  10 cells / ml . the cell 
 suspension ( approximately 2 to 3  10 u937.a2 cells ) 
 was centrifuged at 4 c for 30 min at 1250 rpm to obtain a cell 
 pellet , and then the pelleted cells were lysed in 20 mm tris - hcl , 
 ph 8 , 150 mm nacl , 1% ( mass / volume ) 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate 
 ( chaps ) , 1 mm ( phenylmethylsulfonyl fluoride ) , 10 g / ml pepstatin , 
 5 g / ml aprotinin , and 10 g / ml leupeptin ( sigma - aldrich , 
 st . the mixture was gently rocked in a laboratory shaker 
 for 15 min at 4 c to break up the cells . the cell lysate was 
 subjected to ultracentrifugation at 40,000 rpm for 1 h to remove cell 
 debris . all protein loading and elution 
 steps were then performed using a chemyx fusion 100 syringe pump ( stafford , 
 tx ) . the cell pellet consisted of 2  10 cells 
 of u937.a2 with each cell estimated to have 10,000 hla - a*0201 
 molecules . for efficient isolation of nearly 
 all the hla - a*0201 molecules in the lysate , 100 excess molecules 
 ( 3.3  10 moles ) of the anti - hla - a*0201 antibody , 
 bb7.2 , were introduced into a 5 ml ge healthcare hitrap recombinant 
 protein a column ( chicago , il ) for the immobilization . the bb7.2 antibody 
 molecules were reconstituted in 200 mm tris hcl ( ph 8) ( sigma - aldrich , 
 st . first , the 
 column was washed with 50 mm tris ( ph 8) for 5 min at 1 ml / min flow 
 rate . then , the reconstituted bb7.2 solution ( 1 ml ) was injected into 
 the column at 0.2 ml / min flow rate for 5 min . excess antibody was 
 washed out using 200 mm tris hcl ( ph 8) at 1 ml / min for 5 min . the hla - a*0201/peptide complex containing supernatant was introduced 
 into the bb7.2 antibody immobilized column at 1 ml / min for immunoaffinity 
 capture of hla - a*0201/peptide complexes . then , the column was washed 
 using 50 mm tris ( ph 8) solution at 0.5 ml / min for 5 min to remove 
 excess cell lysate in the column . finally , 0.2 m acetic acid solution 
 ( ph 3 ) was introduced at a flow rate of 0.5 ml / min for 10 min to elute 
 the immobilized hla - a*0201/peptide complexes from the column surface . glacial acetic acid ( 17.5 m original ) was added to the eluate 
 to increase the acetic acid percentage to 10% to dissociate the hla - a*0201 
 from the peptides . finally , the peptides were separated from the high 
 molecular weight proteins by centrifugation at 3220 rpm in 4 c 
 for 40 min on an amicon ultra-3 kda device ( millipore , billerica , 
 ma ) . the lyophilized peptide samples were stored in 80 c 
 until they were analyzed by dims the lyophilized samples obtained from 
 the cell line were reconstituted for electrospray ionization ( esi ) 
 in 60 l of 50/49.9/0.1 acn / h2o / fa ( v / v / v ) and were 
 directly infused for electrospray ionization ( esi ) at a flow rate 
 of 2 l / min . the esi emitter 
 was held at ground potential , and a voltage of 4.25 kv was 
 applied to the custom built planar dims electrode assembly and housing 
 ( figure 1 ) . the two parallel stainless steel 
 4  10 mm electrodes are separated by a 0.3 mm gap . the assembly 
 slides onto the glass transfer capillary of the source region of the 
 mass spectrometer . a housing is then oriented around the assembly 
 to reroute the desolvation gas , which also serves as the carrier gas 
 through the device . the temperature of the desolvation gas is set 
 to 300 c in the instrument software . ideally , a square wave should be used for dims , 
 alternating between 
 low and high electric fields of opposing polarity . however , because 
 of the high power requirements of high - voltage , high - frequency square 
 waves , most dims waveforms are bisinusoidal , approximating a square 
 wave ( figure 2 ) . in these experiments the waveform generator output a sinusoidal 
 wave at 2 mhz and a lower amplitude sinusoidal wave at 4 mhz , phase 
 shifted about 90. each sinusoidal wave is applied to one of 
 the electrodes , and thus , they are capacitatively summed across the 
 gap to form the bisinusoidal dims waveform at frequency of 2 mhz . dims spectra were acquired by scanning the cv 
 using a labview program linked to the instrument control software . ten mass spectra were averaged for each cv in the dims spectra . a 
 static cv for the filter mode can also be selected with the labview 
 program . the ms / ms spectra shown for the filter mode were averaged 
 for 1 min at the static cv . simplified schematic of the addition of sinusoidal 
 waves across 
 the gap to form a bisinusoidal waveform the model peptide 
 pool that was generated was intended to simulate a complex mixture 
 of very similar peptides ( by molecular weight , hydrophobicity , and 
 sequence ) such as that which would be seen in a collection of antigen 
 peptides obtained from a biological source . a dims scan of a solution 
 of the pure  target  peptide , protonated glr ( amino 
 acid sequence = flssanehl ) , v , the optimum cv was determined 
 to be 6.5 v. dims scan of protonated flssanehl ( also called glr m / z 1017.5 ) at a dv of 1200 v. the ms / ms spectrum obtained for pure glr ( m / z 1017.5 ) was compared to that obtained 
 for the mixture 
 of 94 peptides ( figure 4a ) . without glr spiked 
 into the mixture , none of the product ions observed from pure glr 
 were present ( figure 4b ) . the glr peptide was 
 spiked into the 94-peptide pool at concentrations ranging from 1 m 
 to 1 pm . for each solution the ms / ms spectrum at the mass - to - charge 
 ratio of the target protonated glr peptide was taken with and without 
 dims active . the five most intense product ions observed in the ms / ms 
 spectrum of pure glr , [ m + h  h2o ] , 
 [ m + h  nh3 ] , [ m + h  h2o  nh3 ] , b8 , and 
 b8-nh3 were used to evaluate whether glr was 
 detectable at each concentration . with 1 pm glr spiked into the mixture , 
 two of the five product ions were observable at a signal - to - background 
 ratio greater than 3 , where background is defined as the signal obtained 
 for the mixture without glr spiked in . however , the ratio of these 
 product ions deviated too much from the reference spectrum to consider 
 the peptide to be detected at this concentration . with 10 pm glr spiked 
 into the mixture , all five of the product ions were observable , with 
 a contaminant ion present that was observed without glr ( figure 4c ) . without dims , none of the product ions observed 
 at greater than 5% relative intensity match those observed from ms / ms 
 of protonated glr ( figure 4d ) . using the detection 
 of five product ions as indicative of the presence of the target ion 
 ( in typical biomarker detection , only two product ions are used to 
 confirm the presence of an analyte ) , a comparison of the detection 
 of glr in the peptide mix with and without dims was made ( figure 5a , b ) . the red line represents the signal intensity 
 at which the signal - to - background ratio is equal to 3 . in this model 
 peptide pool , the target antigen was readily detected at a concentration 
 of 10 pm in a mixture of 94 other peptides possessing similar chemical 
 properties . each ms / ms scan was obtained using a volume of 2 
 l ; so the system is able to identify peptides at the low attomole 
 level . this level of sensitivity ( 20 amol = 1.2  10 molecules ) is important when considering applying this technique 
 for use in human cancer samples . in the case of acute leukemia , most 
 patient samples have 1  10 leukemia cells 
 per ml of whole blood with each cell possessing 10,000 hla 
 molecules . a routine blood draw of 10 ml would therefore be expected 
 to yield 1  10 peptides , present at various frequencies , 
 making the application of dims ms / ms for leukemia antigen discovery 
 feasible in most clinical samples . in solid tumors , 
 a 1 cm sample contains 1  10 cells ; so again dims ms / ms of protonated 
 flssanehl ( glr ) , where panel a was obtained 
 for 10 m flssanehl in 50/49.9/0.1 acetonitrile / water / formic 
 acid , panels b and c were obtained with dims at a dv of 1200 v and 
 a cv of 6.5 v , where panel b was obtained for a mixture of 94 peptides 
 without flssanehl added , and panel c was obtained for the same mixture 
 of 94 peptides , but with 10 pm flssanehl in the mixture , and panel 
 d was obtained without dims for the mixture of 94 peptides with 10 
 pm flssanehl . sum of the intensities 
 of the five most intense product ions observed 
 from ms / ms of protonated flssanehl as a function of the concentration 
 of flssanehl ( glr ) in the mixture of 94 peptides , where panel a is 
 without dims and panel b is with dims under the optimum conditions 
 for the selection of protonated glr . the red line indicates the signal 
 intensity at which the signal - to - background ratio is 3 , where background 
 is defined as the signal obtained for the mixture of 94 peptides without 
 glr spiked in . a significant problem in cancer immunology 
 is the identification of cancer - specific peptide antigens , which can 
 then be developed into cancer immunotherapeutics , such as vaccines . to demonstrate how dims ms / ms 
 could be applied to this problem , the device was used to detect three 
 known cancer antigens from a model leukemia cell line , u937.a2 . the 
 three peptide antigens targeted , cg1 ( fllptgaea ) , unc - cdk4 - 1 ( altpvvvtl ) , 
 and unc - ankrd17 - 1 ( lliergasl ) , are ( 1 ) derived from proteins that 
 are overexpressed or aberrantly processed in human acute myeloid leukemia 
 ( aml ) , ( 2 ) computationally predicted to bind with high affinity to 
 hla - a*0201 ( the most common hla type ) , and ( 3 ) have been previously 
 shown to be presented by u937.a2 cells . the optimum cv to select for sodiated cg1 ( fllptgaea 
 + na , m / z 940.4 ) at 
 a dv of 1200 v was determined to be 7.8 v from a dims scan of the 
 pure peptide ( figure 6a ) . note that , rather 
 than the analysis being performed with the protonated peptide , the 
 sodiated peptide was used because there was greater sensitivity with 
 the latter . the sodiated peptide would likely not be seen in an lc / ms 
 experiment because the lc effectively  desalts  the 
 sample , and there would be no sodium present in the solvent to effect 
 ionization when the peptide eluted . the ms / ms of the pure cg1 peptide 
 is shown in figure 6b . ms / ms of sodiated peptides 
 has been extensively studied , and in general , the fragmentation efficiency 
 is as good or better than that observed for protonated peptides . with dims , the peptide cell extract ms / ms spectrum of m / z 940.4 exhibited five product ions that matched 
 the ms / ms of the pure peptide as well as a contaminant peak ( figure 6c ) . without dims , none of the product ions observed 
 at greater than 5% relative intensity match those observed from the 
 ms / ms of sodiated cg1 ( figure 6d ) . ( a ) dims scan 
 of sodiated fllptgaea ( m / z 940.6 ) 
 and ms / ms obtained for sodiated fllptgaea , where 
 panel b was obtained for 10 m fllptgaea in 50/49.9/0.1 acetonitrile / water / formic 
 acid , panel c was obtained for the peptide cell extract with dims 
 with a dv of 1200 v and a cv of 7.8 v , and panel d was obtained for 
 the peptide cell extract without dims . this approach was used for two other previously confirmed 
 antigen 
 peptides unc - cdk4 - 1 ( altpvvvtl ) and unc - ankrd17 - 1 ( lliergasl ) . after determining the optimum cvs for unc - cdk4 - 1 
 and unc - ankrd17 - 1 , the same discovery experiment was performed as 
 described above . in the case of unc - cdk4 - 1 , four identifiable product 
 ions could be detected in the ms / ms with dims active and with dims 
 transparent ( supplementary figure s-1 a - c ) . for unc - ankrd17 - 1 , one identifiable product ion could be assigned 
 on the ms / ms with dims active , and no product ions could be identified 
 without dims ( supplementary figure s-2 a - c ) . with one observed product ion , we believe we have detected this 
 peptide but can not confirm its detection due to the lack of additional 
 product ions and inability to perform ms due to low ion 
 intensity . multiple product ions from the ms / ms 
 spectra could be identified with dims active for both cg1 and unc - cdk4 - 1 . many human tumors are susceptible to t - cell 
 mediated killing ; however , 
 to avoid off - target toxicity , t - cell immunotherapeutics such as vaccines 
 or adoptive cellular therapy must target specific cancer - associated 
 antigens . we demonstrate that cancer antigens can be identified 
 using a targeted ms approach , aimed at identifying specific antigens , 
 and that dims significantly improved our ability to confirm the presence 
 of antigens by improving the signal - to - noise of the ms experiments . cancer antigen discovery using nontargeted ms methods have typically 
 required extremely large cell populations of > 1  10 cells per analysis , which makes analysis of most clinical samples 
 impossible . while we originally expanded 
 > 1  10 u937.a2 cells , the isolated peptides were 
 divided into 10 aliquots so that the antigen discovery experiments 
 were performed on peptides from 1  10 cells , 
 a number of cells that can be obtained in many more clinical specimens , 
 particularly aml samples . the ability 
 to detect antigens using these small quantities of cellular material 
 is a result of our targeted approach to antigen discovery and the 
 improved signal - to - noise of the dims device , which we observed in 
 all experiments , when used in a targeted antigen discovery method . over the past two decades , computational algorithms have greatly 
 enhanced our ability to predict cancer antigens . with this advancement , targeted ms approaches , as 
 described in this article , can now be used to screen for and identify 
 high value cancer antigens . this approach is particularly exciting 
 given the dramatic expansion of cancer genome sequencing . with these types of genomic data , it is possible 
 to probe patient tumor genomes and predict candidate antigens that 
 result from genetic lesions in the tumor itself . dims ms / ms could then be applied to specific patient 
 samples to confirm or deny the presence of the predicted antigen , 
 which could lead to more informed decisions regarding possible cancer 
 immunotherapies . modifications to our current device could include 
 further optimization of the dims electrodes to improve focusing of 
 the ion stream in the dims prior to entry into the ms and the use 
 of nanospray ionization .
differential ion mobility spectrometry 
 ( dims ) can be used as a 
 filter to remove undesired background ions from reaching the mass 
 spectrometer . 
 the ability to use dims as a filter for known analytes 
 makes dims coupled to tandem mass spectrometry ( dims  ms / ms ) 
 a promising technique for the detection of cancer antigens that can 
 be predicted by computational algorithms . in experiments using dims 
 ms / ms 
 that were performed without the use of high - performance liquid chromatography 
 ( hplc ) , a predicted model antigen , glr ( flssanehl ) , was detected at 
 a concentration of 10 pm ( 20 amol ) in a mixture containing 94 competing 
 model peptide antigens , each at a concentration of 1 m . without 
 dims filtering , the glr peptide was undetectable in the mixture even 
 at 100 nm . 
 again , without using hplc , dims 
 ms / ms was used to 
 detect 2 of 3 previously characterized antigens produced by the leukemia 
 cell line u937.a2 . 
 because of its sensitivity , a targeted dims 
 ms / ms 
 methodology can likely be used to probe for predicted cancer antigens 
 from cancer cell lines as well as human tumor samples .