in eukaryotes , the basic repeating unit 
 of chromatin is the nucleosome , consisting of 147 base pairs of dna 
 wrapped around an octamer of histone proteins . the octamer contains 
 two copies of each of the core histones , h2a , h2b , h3 , and h4 . histone proteins are dynamically post - translationally 
 modified to regulate a wide array of nuclear processes , including 
 transcription , dna damage repair , and signaling pathways . most of this post - translational modification 
 ( ptm ) occurs on the n - terminal histone tails that protrude from the 
 nucleosomal surface . of the core histones , 
 h3 and h4 undergo the most extensive modifications mass spectrometry 
 ( ms ) has emerged as a powerful tool to characterize histone ptms as 
 it can accurately identify and quantify all histone ptm profiles in 
 an unbiased manner . ms can also identify novel modifications 
 as well as multiple modifications on a single peptide . most histone ptm analysis by ms has been conducted on high - resolution 
 mass analyzers , such as orbitraps , because they provide sufficient 
 mass accuracy to differentiate two very common , nearly isobaric ptms : 
 acetylation and trimethylation , 42.0106 and 42.0470 da , respectively . for example , the ultrahigh - resolution fourier transform ion cyclotron 
 resonance mass spectrometer ( fticr  ms ) has a resolving power 
 ( > 10 ) that can differentiate peptides with almost identical 
 mass ( m / z < 0.001 ) , and 
 can therefore easily discriminate trimethylation and acetylation based 
 on mass alone . lower - resolution mass analyzers , 
 such as linear ion traps , are unable to resolve this small mass difference . however , other techniques can be used to 
 differentiate these modifications on lower - resolution mass analyzers , 
 such as ms / ms spectra in combination with retention time information or synthetic peptide coelution information . the hunt lab demonstrated that the h3 917 aa peptide bearing 
 k14ac or k9me3 eluted 17 min apart based on a 240 min gradient ( 0 
 to 100% b ) , suggesting that relative retention time can be used to 
 distinguish the modifications . subsequently , 
 the freitas group utilized this shift in retention time to demonstrate 
 that trimethylated and acetylated peptides can be distinguished on 
 low - resolution mass spectrometers based on their relative retention 
 times . they validated their peak assignments by running the same sample 
 on a high - resolution mass spectrometer to resolve the mass difference 
 on the ms1 level and also performed ms / ms for further validation . one way to accomplish this 
 task is to use heavy isotope labeled methyl and/or acetyl donors so 
 that the modifications are no longer isobaric . however , it is relatively time - consuming 
 to wait for the light modifications to turn over . another option is 
 to spike in heavy labeled synthetic peptides , which will then coelute 
 with the modified peptide of interest , but will be distinguishable 
 based on its unique mass . used synthetic peptide coelution 
 to determine that a potential new modification on a histone peptide 
 ( h2at15ac ) was falsely assigned . neutral losses can also be used to lend confidence to identity assignments 
 for isobaric peptides . trimethylated peptides undergo signature neutral 
 losses during collision induced dissociation ( cid ) fragmentation , 
 and the resulting mass shifts observed in ms / ms spectra can be used 
 to validate methylation sites . fragmentation of acetylated 
 peptides does not result in neutral losses but does produce immonium 
 ions at m / z 143.1 which can be used 
 to verify the presence of acetylated resides . since lower - resolution instruments are less expensive , easier 
 to maintain , and somewhat more ubiquitous compared to high - resolution 
 instruments , we aimed to determine if lower - resolution mass analyzers 
 are as capable of robust and accurate histone ptm identification and 
 quantification as high - resolution mass analyzers using several of 
 the above - described techniques . to this end , we compared the performance 
 of a low - resolution linear ion trap ( ltq velos pro ) and a high - resolution 
 ion trap  orbitrap hybrid instrument ( ltq - orbitrap velos pro ) 
 in comprehensive ptm analysis of histones , one of the most extensively 
 modified proteins in eurkaryotes . most modifications have a large 
 enough mass difference to be resolved on lower - resolution mass analyzers , 
 and so one of the biggest challenges when using low - resolution detectors 
 for histone ptm analysis is differentiating trimethylation from acetylation . we first determined the optimal scan mode on the ltq velos pro 
 based on three criteria : ( 1 ) reproducibility of relative peptide abundance 
 measurements , ( 2 ) resolution of peptides in higher charge states ( e.g. , 
 + 2 and + 3 charges ) , and ( 3 ) the number of ms1 and ms2 scans obtained 
 per run . from this analysis , we determined that enhanced scan mode 
 has the optimal trade - off between resolution and number of scans , 
 while providing very consistent quantification . we then compared the 
 performance of the ltq velos pro on enhanced scan mode to the orbitrap 
 velos pro using the same criteria listed above as well as mass accuracy . the orbitrap velos pro was able to resolve the mass difference between 
 acetylation and trimethylation , while the ltq velos pro could not . however , these modifications could be accurately quantified on the 
 ltq velos pro using either retention time information or stable isotope - labeled 
 synthetic peptide spike - in standards . as such , we conclude that while 
 high - resolution mass analyzers are ideally suited for histone ptm 
 analysis , low - resolution detectors are also adequate . hela s3 cells were gown in suspension as described previously and harvested using standard protocols . nuclei were resuspended in 0.4 m nacl 
 buffer containing 1 mm dtt , 0.3 mm aebsf , and 10 mm sodium butyrate 
 ( 10:1 buffer : pellet by volume ) and were incubated at 4 c with 
 shaking for 30 min . the nuclei were pelleted at 3000 g for 5 min at 4 c , and the supernatant was removed by decanting . the pellet was resuspended in 2.5 m nacl containing 1 mm dtt , 0.3 
 mm aebsf , and 10 mm sodium butyrate ( 5:1 by volume ) . an equal volume 
 of cold 0.4 n h2so4 was added slowly , and the 
 nuclei were incubated at 4 c with shaking for 2 h. the nuclei 
 were pelleted at 3400 g for 5 min , and proteins were 
 precipitated from the supernatant with tca as previously described . when needed , offline reverse - phase high - performance 
 liquid chromatography ( rp - hplc ) was used to further purify histone 
 proteins as described previously . acid - extracted total histones or rp - hplc - purified 
 histones were chemically derivatized with propionic anhydride and 
 digested using trypsin ( promega ; substrate / protease ratio of 20:1 ) 
 as described previously . histone peptides 
 were subsequently desalted for ms analysis using homemade c18 stage 
 tips as described previously . briefly , 93 synthetic tryptic histone peptides containing 
 heavy - labeled amino acids were synthesized containing the most common 
 ptm profiles . the peptides were purified by rp - hplc and combined to 
 a final concentration of 0.27 pmol/l / peptide in water . the synthetic peptide library was added to a 
 total acid - extracted histone sample in a ratio of 100 fmol of synthetic 
 peptides:1 g of histone . a fused silica microcapillary column ( 75 m i.d . ) was packed 
 in - house with reprosil - pur c18 resin ( 3 m , dr . the column was fitted to a commercial fused silica emitter with a 
 10 m tip ( new objective ) . small aliquots ( 1.5 g ) of 
 a single histone h4 sample were loaded onto the column by an eksigent 
 nanolc as-2 autosampler . peptides were separated using an eksigent 
 nanolc 2d plus system hplc across a 76 min multistep gradient : 2% 
 buffer b for 1 min ( a , 0.1% formic acid in water ; b , 0.1% formic acid 
 in acetonitrile ) , 2% to 30% b in 55 min , 30% to 98% b in 15 min , 98% 
 b for 10 min , 98% b to 2% b in 30 s , 2% b for 9.5 min ( equilibration ) 
 at a constant flow of 250 nl / min . the hplc was coupled to a linear 
 quadrupole ion trap ( ltq velos pro , thermo scientific ) mass spectrometer 
 operating in zoom , enhanced , normal , or turbo scan modes ( see table 1 for scan rate information ) . a full ms spectrum 
 was obtained followed by 6 data - dependent ms / ms acquisitions for the 
 top six most abundant ions using cid fragmentation ( collision energy 
 of 40 , activation q of 0.25 , and activation time 
 of 10 ms ) . three technical replicates were obtained for each scan 
 mode . based on a 76 min 
 gradient ( 2% b for 1 min ; 2% to 30% b in 55 min ; 30 to 98% b in 15 
 min ; 98% b for 10 min ; 98% to 2% b in 30 s ; 2% b for 9.5 min ) at 250 
 nl / min flow rate using an eksigent nanolc ultra loading pump . aliquots ( 1.5 g ) of the 
 same acid - extracted histone sample with synthetic peptides were run 
 on two separate mass spectrometers : a linear ion trap ( ltq velos pro ) 
 and a hybrid linear ion trap column was used on both instruments ( packed 
 in - house with 3 m c18 resin ) fitted with a fused silica emitter 
 with a 10 m tip ( new objective ) . the sample was loaded using 
 an eksigent nanolc 2d plus system hplc for the ltq instrument while 
 a thermo easy nlc 1000 was used to load the samples for the hybrid 
 ltq - orbitrap instrument . the same multistep gradient was used for 
 both instruments , consisting of 2% b for 1 min , 2 to 30% b in 40 min , 
 30% to 98% b in 15 min , 98% b for 10 min , with the exception that 
 the eksigent nanolc gradient had an additional 10 min equilibration 
 step at 2% b. data acquisition for both instruments was broken into 
 3 segments , 14 , 26 , and 16 min long , respectively . in the first segment , 
 a full ms was obtained followed by 9 data dependent ms / ms acquisitions 
 of the top nine most abundant ions from the full ms . in the second 
 segment , a full ms was obtained , followed by 5 targeted ms / ms acquisitions 
 ( m / z : 528.30 , 570.84 , 754.93 , 761.94 , 
 and 768.95 ) and 5 data dependent ms / ms acquisitions of the top 5 most 
 abundant ions from the full ms . in the third segment , a full ms was 
 acquired followed by 10 data dependent ms / ms acquisitions of the top 
 10 most abundant ions . all full ms acquisitions for the orbitrap velos 
 pro were obtained in the orbitrap in profile mode ( resolution : 60,000 
 at m / z 400 ) , while ms / ms acquisitions 
 were obtained in the ion trap . cid fragmentation was used to fragment 
 ions ( collision energy of 40 , activation q of 0.25 , 
 activation time of 10 ms ) . we used an in - house 
 developed algorithm to identify and quantify ptms for the ltq - orbitrap 
 velos pro data , as described in wu et al . the algorithm uses ms , ms / ms , and retention time information to 
 accurately identify and provide relative abundances of ptm profiles 
 for tryptic histone peptides . relative abundance is obtained by integrating 
 the single ion chromatogram of a particular modified peptide in all 
 occupied charge states and dividing it by the total ion current for 
 that peptide in all of its modified forms . the relative abundances 
 of three technical replicates were averaged to obtain the final estimation 
 of relative abundance . all ltq data were manually quantified because 
 they are not compatible with the aforementioned algorithm . monoisotopic 
 raw abundance of each peptide was manually extracted from xcalibur 
 qual browser , based on the area underneath extracted ion chromatograms 
 at the full ms level . all detectable charge states were considered , 
 typically [ m + h ] , [ m + 2h ] , and [ m + 3h ] as previously described . the 
 ltq velos pro mass spectrometer can operate in four different scan 
 modes that have varying scan rates : turbo , normal , enhanced , and zoom 
 ( rates listed in table 1 ) . the scan rate affects 
 several important properties , including reproducibility of peptide 
 abundance measurements , resolution , and number of scans collected 
 per run . the optimal scan mode will vary depending on the specific 
 application , and , as such , we sought to determine which scan mode 
 is optimal for histone ptm analysis based on their ability to resolve 
 doubly and triply charged histone peptides while still allowing for 
 the maximal number of scans and consistent quantification . to our 
 knowledge , this study represents the first comprehensive analysis 
 of different scan modes for histone ptm analysis on a low - resolution 
 mass spectrometer . three technical replicates of a single histone 
 h4 sample were analyzed for this experiment . the histone sample was 
 purified from hela cells treated with butyrate , a histone deacetylase 
 inhibitor , therefore containing more pronounced and combinatorial 
 acetylation profiles . the sample was derivatized using our propionic 
 anhydride methodology as it increases the hydrophobicity of the histone 
 peptides , allowing for better retention on c18 resin , and also prevents 
 trypsin cleavage after lysine . to reduce variance due to instrument 
 setup , the sample replicates were run using identical chromatographic 
 gradients ( delivered by different hplcs ) and the same analytical column 
 within 2 days . since small changes in ptm abundances can have 
 large biological implications , it is important to run samples for 
 ptm analysis on an instrument that generates highly reproducible results . 
 therefore , any observed differences can be attributed to biological 
 phenomena rather than technical variances . as such , we investigated 
 the reproducibility of measuring the relative abundances of modifications 
 for a typical example peptide from histone h4 ( amino acids 417 ; 
 sequence : gkggkglgkggakr ) , for each scan mode . relative 
 abundances were obtained by integrating the single ion chromatogram 
 ( sic ) in ms1 spectra of a particularly modified peptide in all occupied 
 charge states and normalizing the signal by dividing it by the total 
 ion current of that peptide in all of its modified forms , as we and 
 others have performed for many years now . the 
 relative abundance values obtained on each scan mode were found to 
 have a fairly small standard deviation , indicating that each mode 
 produces highly reproducible abundance measurements ( figure 1 ) . however , some scan modes produce different relative 
 peptide abundance measurements compared to the other scan modes , with 
 turbo scan mode being the most variable . this variability indicates 
 that turbo scan mode may lead to inaccuracies in abundance measurements , 
 possibly due to the presence of multiple species in a given m / z range that can not be resolved . the 
 tri- and tetra - acetylated peptides have larger standard deviations 
 than the other modified forms of the peptide , which is more common 
 for low - abundance peptides . reproducibility of relative peptide abundance 
 measurements for h4(417aa ) peptides on each ltq velos pro 
 scan mode . the relative abundance of each modified form of the peptide 
 was calculated from three technical replicates of the same purified 
 h4 sample . error bars represent standard deviation from average relative 
 peptide abundances . * p < 0.05 . both resolution and scan rate are important factors 
 affecting accuracy in peptide and ptm identification . obtaining adequate 
 resolution is vital for identifying and quantifying the ptm profiles 
 present in a sample . if the resolution is too low , the charge state 
 of the peptides can not be determined , and the identity can not be confidently 
 assigned . ion trap mass analyzers scan and record the m / z range by increasing the radio frequency ( rf ) voltage applied 
 to the electrode over time to eject peptide ions of increasing m / z from the trap to be detected . fast 
 scan rates result in poor resolution because the ions of a given m / z do not have enough time to fully eject 
 before the rf increases , causing peak widening . the resolution obtained in the four different ltq 
 velos pro scan modes is shown in figure 2a 
 for an example peptide , diacetylated h4 417 + 2 ( [ m + 2h ] = 761.939 ) . the fastest scan rate , turbo , is completely 
 unresolved , and the isotopes can not be distinguished at all . however , 
 the zoom , enhanced , and normal scan modes have sufficient resolution 
 to reliably assign charge states and are therefore amenable to this 
 type of analysis . resolution of diacetylated h4 417 on each ltq 
 velos pro scan mode . mass spectra of ( a ) doubly charged ( [ m + 2h ] = 761.939 ) and ( b ) triply charged ( [ m + 3h ] = 508.295 ) peptides are displayed . the resolution ( m / m ) of the monoisotopic peak ( c ) resolution of the peptides from panels a and b as a function of 
 scan rate . as the charge state increases , 
 it becomes more difficult to resolve the isotopes of a given peptide . propionylated tryptic histone peptides generally occupy lower charge 
 states ( + 1 to + 3 ) . figure 2b displays the resolution 
 of the triply charged example peptide ( [ m + 3h ] = 508.295 ) . zoom and enhanced modes can resolve the charge state , while normal 
 and turbo scan modes do not fully resolve the peptide . therefore , 
 zoom and enhanced scan modes are better options for histone ptm analysis 
 on a low - resolution mass spectrometer . figure 2c illustrates the relationship between scan rate and resolution for 
 the example peptide in both charge states . one important note 
 is that scan rate affects the amount of data that can be collected 
 during an ms analysis . zoom has the best resolution , but is also the 
 slowest scan rate and therefore does not collect as many full ms and 
 ms / ms spectra as enhanced scan mode ( table 1 ) . since some modified peptides have low abundances ( e.g. , k18me1 , 
 k23me1 ) , it may be advantageous to sacrifice higher resolution for 
 an increased number of scans . as such , we recommend using enhanced 
 mode for analyzing histone ptms as it has sufficient resolution to 
 resolve isotopes for double and triply charged peptides , allows reproducible 
 quantification , and allows for almost 20% more ms2 scans compared 
 to zoom mode . high - resolution mass analyzers have been at the forefront in histone 
 ptm analysis , despite the fact that low - resolution mass analyzers 
 are less expensive and generally more accessible . one of the main 
 reasons for using a high - resolution detector is that it can resolve 
 the mass difference between trimethylation and acetylation modifications 
 ( 42.0470 and 42.0106 da , respectively ) , which occur at high frequency 
 on histone proteins . however , it is still possible to differentiate 
 these ptms on a low - resolution detector using a combination of retention 
 time and ms / ms information . furthermore , 
 krey et al . demonstrated that the low - resolution ltq and ltq velos 
 pro mass spectrometers can quantify protein abundance as accurately 
 as high - resolution orbitrap mass spectrometers over a range of protein 
 concentrations . as such , low - resolution 
 mass analyzers could potentially be useful in analyzing histone ptm 
 profiles , however to our knowledge no side - by - side comparison between 
 low- and high - resolution mass analyzers has been published yet for 
 comprehensive analysis of histones , which contain one of the most 
 complicated ptm profiles of any eukaryotic protein . we therefore compared 
 the performance of the ltq and orbitrap mass analyzers to determine 
 if low - resolution mass analyzers are adequate for this type of complicated 
 analysis . to conduct this comparison , we used a single sample 
 containing total acid - extracted histones from butyrate - treated hela 
 cells , which were subsequently propionylated and digested with trypsin . 
 propionylated , heavy isotope - labeled synthetic peptides were spiked 
 in the hela histone peptides to aid in identification of endogenous 
 histone peptides during data analysis , as will be described in more 
 detail later . we analyzed three technical replicates of this sample 
 on each instrument and compared the resulting reproducibility of relative 
 peptide abundance values , resolution , and mass accuracy . the ltq velos 
 pro was operated in enhanced scan mode as this was determined to be 
 optimal for histone ptm analysis . the runs were conducted within 3 
 days on the same analytical column , instrument method , and gradient 
 to reduce differences due to instrument setup . one drawback 
 of using the orbitrap velos pro mass analyzer is that the scan rate 
 is inherently slower than that of the ltq velos pro mass analyzer . table 2 compares scan rate information for 
 the ltq velos pro and the orbitrap velos pro . the scan numbers refer 
 to the number of scans collected in the first three segments ( see experimental section ) . the orbitrap velos pro collected 
 an average of 873 full ms scans and 8,784 ms / ms scans , while the ltq 
 velos pro collected 1,189 and 11,703 , respectively ( table 2 ) . thus , while high - resolution detectors allow 
 for better mass accuracy and consequently more facilitated data analysis , 
 low - resolution detectors collect a larger amount of information even 
 when operated on one of the slowest scan modes . this trade - off of 
 reduced accuracy for increased ms and ms / ms scans could be desirable 
 for the identification and quantification of rare or low - level ptms . the orbitrap data is based on a 66 min gradient that omits the 
 final equilibration step ( 2% b for 1 min ; 2% to 30% b in 55 min ; 30 
 to 98% b in 15 min ; 98% b for 10 min ) at 250 nl / min flow rate using 
 a thermo easy nanolc hplc . as mentioned previously , it is important that the instrument used 
 for ptm analysis generate highly reproducible abundance measurements . 
 as such , we compared the reproducibility of calculated relative peptide 
 abundances obtained on the ltq velos pro and the orbitrap velos pro 
 instruments based on three technical replicates each . we only analyzed 
 h3 and h4 because they are the most modified histone proteins and 
 are therefore the most challenging to analyze . the results , as shown 
 in figure 3 , demonstrate that the standard 
 deviation in relative abundance measurements is very small on both 
 instruments , and results are very comparable between both instruments 
 ( p = 0.09 ; paired t test comparing 
 standard deviations between the two instruments ) , indicating that 
 both instruments are highly reproducible . reproducibility 
 of relative peptide abundance measurements obtained on the ltq velos 
 pro and the orbitrap velos pro . ( a , b ) each point represents a particular 
 modified form of either ( a ) an h3 peptide or ( b ) an h4 peptide , while 
 the color of the point indicates the identity of the peptide . the 
 black line indicates perfect correlation between relative peptide 
 abundance values on the two instruments . the dashed line is a linear 
 regression for all data points on the plot ( pearson , ( a ) r = 0.878 , ( b ) r = 0.839 ; 
 spearman , ( a ) r = 0.853 , ( b ) r = 0.883 ) . the modified peptides shown in the 
 figure include the following : ( a ) 38 , unmodified , k4me1 ; 917 , 
 unmodified , k9me1 , k9me1k14ac , k9me3k14ac , k9me2 , k9me3 , k9 or k14ac , 
 k9me2k14ac ; 1826 , unmodified , k18ac or k23ac ; 2740 , 
 unmodified , k36me1 , k36me2 , k27me1 , k27me2 , k27me3 , k27me2k36me1 , 
 k27me1k36me2 , k27me1k36me3 , k27me1k36me1 ; ( b ) 417 , unmodified , 
 monoacetylated , and diacetylated ; 2023 , unmodified , k20me1 , 
 k20me2 . each point represents a particular 
 modified form of the h3 917 peptide , listed in panel b. the 
 blue points represent the data before normalization as shown in panel 
 b , and the red points represent the data after normalization to the 
 synthetic peptide standards . the dashed blue line is a linear regression fit of the 
 data before normalization ( r = 0.579 ) , 
 and the red dotted line is a linear regression fit of the data after 
 normalization ( r = 0.979 ) . since the same sample was analyzed on both instruments , 
 we would expect the calculated relative abundance measurements on 
 each instrument to be similar . the results indicate that while in 
 general this is true , some of these values differ between the two 
 instruments ( figure 3a , b , as evidenced by deviation 
 from the black line that indicates perfect correlation ) . however , 
 a linear regression fit to all data points for h3 or h4 peptides demonstrates 
 a high degree of correlation between the values obtained on each instrument 
 ( pearson , r = 0.878 for h3 and r = 0.834 for h4 ; spearman , r = 0.853 for h3 and r = 
 0.883 for h4 ) , indicating that the instruments produce highly similar 
 abundance measurements . it is also important to note that the measurements 
 are relative , so an error in the abundance of one modified form of 
 a peptide affects the relative abundance measurement of all other 
 forms of that peptide due to the way the data is normalized . although the correlation of peptide abundance measurements between 
 the two instruments is relatively high overall , some peptides do not 
 correlate well . the peptide with the lowest correlation between instruments 
 was the h3 917 peptide ( pearson : r = 0.579 ) . low correlation is likely a result of the major differences 
 in how the instruments collect data . there were no common features 
 between modifications or peptides that did not correlate well . to 
 correct for differences in instrument data acquisition , the relative 
 peptide abundance measurements of the endogenous peptides can be normalized 
 to those of the synthetic peptide standards , which are present in 
 equal concentrations . to normalize the data , a normalization factor 
 was calculated for each form of the peptide by dividing the expected 
 relative abundance measurements of the synthetic peptides by the observed 
 relative abundance measurement of the synthetic peptides . the average 
 raw abundance values for the endogenous peptides were then multiplied 
 by the respective normalization factor , and the relative peptide abundances 
 were calculated from these corrected values . our group has previously 
 used this method to correct for differences in ionization efficiencies 
 between differently modified histone peptides during quantification . figure 3c plots the relative 
 abundances of the h3 917 peptides before and after normalization 
 to the synthetic peptide standards . after normalization , the abundance 
 measurements obtained on the two instruments are very highly correlated 
 ( pearson : r = 0.979 ) , indicating that 
 the ltq velos pro measures histone ptm abundances as accurately as 
 the orbitrap velos pro . we calculated the average mass accuracy 
 of the ltq velos pro and the orbitrap velos pro based on two example 
 peptides , h3 1826 and h4 417 , in different modified 
 forms to gauge what mass tolerance can be used to identify peptides . 
 the mass accuracy for the ltq velos pro ranges from 53.0 to 193 ppm , 
 with an average of 129 ppm , and the mass accuracy for the orbitrap 
 velos pro ranges from 0.00 to 6.62 ppm , with an average of 2.01 ppm 
 ( table 3 ) . using a mass tolerance of 10 ppm , 
 the orbitrap velos pro can differentiate trimethylation and acetylation , 
 while the ltq velos pro , using a mass tolerance of 150 ppm , can not , 
 as has been noted by others . this is demonstrated 
 in figure 4 , which shows the chromatograms 
 of different modified forms of the histone h3 917 peptide . 
 for the orbitrap velos pro , the trimethylated and acetylated peptide 
 ( row 4 and 5 , respectively ) have a large enough mass difference under 
 the given mass tolerance to be unequivocally assigned to the single 
 peak , while the ltq velos pro can not resolve these peptides . the resolution 
 of the ltq velos pro is high enough to differentiate all of the other 
 common ptms , such as mono- and dimethylation and multiply modified 
 peptides such as k9me1k14ac ( figure 4 , rows 
 13 ) . chromatographic information 
 for h3 917 obtained on ltq velos pro or ltq - orbitrap velos 
 pro . the mass tolerance used for selection is 150 ppm for the ltq 
 velos pro and 10 ppm for the ltq - orbitrap velos pro . in each row , 
 labeled 1 to 7 , the mass of the peptide bearing a specific modification 
 was specified . rows 1 to 5 represent endogenous peptides while rows 
 6 and 7 represent heavy - labeled synthetic peptides . the acetylated and trimethylated peptides can still 
 be distinguished on the ltq , however , by using relative retention 
 time information and high - resolution ms / ms data as has been noted 
 by the freitas group . however , this group 
 did not derivatize their samples with propionic anhydride and did 
 not use this technique to quantify peptides , and so we decided to 
 apply this method to quantify histone ptms for the instrument comparison . synthetic peptides eliminate the need to perform high - resolution ms 
 to validate peak assignments . the retention time of acetylated peptides 
 is later than their corresponding methylated peptides because acetyl 
 groups are more hydrophobic than trimethyl groups ( figure 4 , row 4 versus 5 ) . since 
 the peptides are propionylated in this study , the di- and trimethylated 
 peptides are more hydrophilic than the unmodified peptide . di- and 
 trimethylation prevent propionylation so k9 is propionylated in the 
 unmodified peptide and is not propionylated in the di- and trimethylated 
 peptides . since propionyl groups are more hydrophobic than di- and 
 trimethylation , the unmodified peptide is more hydrophobic and elutes 
 later ( figure 4 , row 3 and 4 compared to 1 ) . heavy - labeled synthetic peptides bearing a trimethyl or acetyl 
 group at the residue of interest can also be used to validate peak 
 assignments . similar methods have been described by others , but there 
 have been no studies using heavy - labeled synthetic peptides to differentiate 
 nearly isobaric ptms . the synthetic peptides have the same retention time as the unlabeled 
 endogenous histone peptides containing the same modifications , as c or n isotopes do not influence retention on 
 c18 columns , but impart a unique mass . therefore , we can determine which ambiguous peak contains a trimethylated 
 or acetylated peptide by seeing which coelutes with the synthetic 
 peptide bearing the respective modification . in figure 4 , synthetic peptides were used to differentiate trimethylated 
 and acetylated peptides . rows 6 and 7 show the chromatograms for the 
 h3 917 synthetic peptides bearing k9me3 or k14ac , respectively . 
 the k14ac synthetic peptide ( row 7 ) elutes under the k14ac peptide 
 peak in the endogenous trace ( row 5 ) , and the k9me3 synthetic peptide 
 ( row 6 ) elutes under the corresponding k9me3 peak in the endogenous 
 trace ( row 4 ) . therefore , low - resolution detectors can be used to 
 accurately quantify and identify histone ptm profiles when supplemented 
 with synthetic peptides . the results of the current study show 
 for the first time that low - resolution mass analyzers , such as the 
 ltq velos pro , are adequate for comprehensive ptm analysis although 
 the data analysis is more easily facilitated on high - resolution instruments . the reproducibility of obtained relative peptide abundances is equally 
 high on the ltq velos pro as the orbitrap velos pro . although the 
 ltq velos pro is not able to resolve the small mass difference between 
 acetylation and trimethylation , other orthogonal lines of evidence 
 can be used to accurately identify the peak containing each modified 
 peptide . as such , high - resolution does not seem to be absolutely required 
 for histone ptm analysis .