protein phosphorylation 
 can modulate protein activity , turnover , subcellular localization , 
 complex formation , folding and degradation .
dynamic phosphorylation 
 plays a pivotal role in almost all biological processes including 
 cell division , differentiation , polarization and apoptosis .
the importance of 
 this post - translational modification ( ptm ) for cell biology has driven 
 the development of novel mass spectrometric tools for sensitive and 
 global detection of phosphorylation . however , the 
 analysis of phosphorylated peptides by mass spectrometry is still 
 not as straightforward as for  regular  , unmodified 
 peptides .
one of the major challenges in phosphoproteomics is to improve 
 ms level representation since phosphopeptides are usually present 
 at substoichiometric levels .
hence , an enrichment step is necessary 
 to enable deeper penetration of the phosphoproteome .
enrichment is 
 typically performed by chromatography , antibodies or metal - ion / metal oxide 
 affinity - based techniques .
two other main challenges 
 are the identification of phosphopeptides and confident localization 
 of the corresponding phosphosite .
the 
 challenge is caused by the higher lability of the phosphate group 
 when compared to the amide bond .
a number of strategies have been 
 proposed to circumvent poor fragmentation and improve sequence and 
 site diagnostic fragmentation , including the use of neutral loss - triggered 
 ms / ms / ms and multistage activation ( msa ) in ion traps , the use of beam type cid fragmentation , and electron capture / transfer dissociation or a combination of some of these approaches .
once phosphopeptide identification is feasible through sufficient 
 peptide backbone fragments , it can still be challenging to pinpoint 
 the true phosphosite .
this becomes more difficult as the number of 
 potential phosphorylation sites within the peptide sequence increases . 
 in principle
direct validation is feasible 
 through detection of a fragment ion that carries the phosphate group . 

neutral loss fragment ions can be used as well ; however , since they 
 exhibit the same mass as a water loss from an unmodified residue they 
 do not directly confirm the correct site .
diagnostic phosphosite - specific fragments facilitate pinpointing 
 the correct phosphosite . several algorithms and programs 

these software tools are based on distinct but similar approaches 
 and they all aim to provide a metric that allows for assessment of 
 the confidence in phosphosite localization .
recently , taus et al . 
 have reported on a new algorithm , coined phosphors , which presently is uniquely compatible with cid , hcd and 
 etd fragmentation and was optimized for both low- and high - resolution 
 ms / ms spectra .
generally , 
 all scoring tools depend on the quality of the ms / ms 
 spectra .
the more site - determining ions are detected , the higher the 
 confidence in phosphosite localization .
we have recently introduced 
 a novel fragmentation scheme combining electron - transfer and higher - energy 
 collision dissociation , termed ethcd .
this method employs dual fragmentation to generate both b / y and 
 c / z ions which leads to very fragment ion- and thus data - rich ms / ms 
 spectra . compared to hcd and etd , we found a substantial increase 
 in peptide backbone fragmentation , which translated into a remarkable 
 average peptide sequence coverage of 94% for tryptic peptides . 

we reasoned that localization of post - translational modifications 
 could also highly benefit from ethcd spectra .
here , we systematically 
 assessed the impact on phosphosite localization using ethcd . in this 
 work
we evaluate the performance of ethcd in comparison to etd and 
 hcd using a defined synthetic phosphopeptide mixture and also on a 
 larger data set of ti - imac enriched phosphopeptides , 
 all in combination with a modified version of phosphors .
all chemicals were purchased from sigma - aldrich 
 ( steinheim , germany ) unless otherwise stated .
protein from hela cells was harvested 
 and digested with trypsin , as previously described .
briefly , gel - loader tips that were plugged with 
 c8 material ( 3 m , zoeterwoude , the netherlands ) were filled up to 1 
 cm with ti - imac beads .
peptides were reconstituted in loading 
 buffer , loaded onto the columns and washed with washing buffer 1 ( 50% 
 acn , 0.5% tfa , 200 mm nacl ) and subsequently washing buffer 2 ( 50% 
 acn , 0.1% tfa ) .
phosphopeptides were eluted with elution buffer 1 
 ( 10% nh3 in h20 ) followed by elution buffer 
 2 ( 80% acn , 2% fa ) .
eluate was acidified and diluted with formic acid 
 to a final acetonitrile concentration of < 5% , split into three 
 equal amounts and directly analyzed by single run lc
all data was acquired on an etd enabled 
 thermo scientific ltq orbitrap velos mass spectrometer ( thermo fisher 
 scientific , bremen , germany ) . a thermo scientific easy - nlc 1000 ( thermo 
 fisher scientific , odense , denmark ) was connected to the ltq orbitrap 
 velos mass spectrometer .
briefly , all spectra 
 were acquired in the orbitrap at a resolution of 7500 .
hcd normalized collision energy was set to 30% for ethcd 
 ( calculation based on precursor m / z and charge state ) .
peak lists were generated using thermo 
 scientific proteome discoverer 1.3 software ( thermo fisher scientific , 
 bremen , germany ) .
the nonfragment filter was used to simplify etd 
 spectra with the following settings : the precursor peak was removed 
 within a 4 da window , charged reduced precursors were removed within 
 a 2 da window , and neutral losses from charge reduced precursors were 
 removed within a 2 da window ( the maximum neutral loss mass was set 
 to 120 da ) .
ms / ms spectra were searched against a database containing 
 the synthetic phosphopeptide sequences and the human uniprot database 
 ( version v201012 ) , respectively , including a list of common 
 contaminants using sequest or mascot ( matrix science , uk ) .
the precursor 
 mass tolerance was set to 10 ppm , the fragment ion mass tolerance 
 was set to 0.02 da .
oxidation of methionine and 
 phosphorylation ( s , t , y ) were used as variable modification and carbamidomethylation 
 of cysteines was set as fixed modification .
percolator was used to filter the psms for < 1% false - discovery - rate . 

phosphorylation sites were localized by applying a custom version 
 of phosphors ( v3.0  ethcd enabled ) 
 that has been expanded to allow analysis of ethcd data .
briefly , the algorithm considers both hcd- and 
 etd - type fragment ions at the same time . while singly and doubly charged 
 b- and y - type fragment ions including neutral loss of phosphoric acid 
 ( h3po4 ) are considered for site localization , 
 only singly charged c- , z - radical and z - prime ions are scored .
observing 
 all possible peptide backbone cleavages in a single ms / ms spectrum 
 substantially simplifies phosphosite localization .
recently , we showed 
 that ethcd enables complete peptide sequencing through dual fragmentation . in ethcd ,
the peptide precursor is initially 
 subjected to an ion / ion reaction with fluoranthene anions in a linear 
 ion trap , which generates c- and z - ions .
however , the unreacted precursor 
 and the charge - reduced precursor remain highly abundant after etd . 
 in the second step
this generates b- and y - ions from the unreacted precursor 
 and simultaneously increases the yield of c- and z - ions by fragmentation 
 of the charge reduced precursor . since the remaining unreacted precursor 
 population is higher charged than the etd - derived fragment ions one 
 can apply a level of energy that fragments the precursor but does 
 not induce secondary fragmentation of c- and z - ions . here
, we continue 
 to explore the benefits of this novel fragmentation mode for the analysis 
 of phosphopeptides . to evaluate the potential added value 
 of phosphopeptide analysis by ethcd we initially used a defined mixture 
 of well - characterized synthetic phosphopeptides .
this mixture consists 
 of 30 phosphopeptides of varying length with up to four phosphorylated 
 residues ( see supplementary table 1 for 
 a complete list , supporting information ) .
we used identical instrument 
 settings with the only exception being the parameters for peptide 
 dissociation , which were set to the for each method optimized values . 

the data was searched with sequest and the psms were manually validated 
 and filtered ( 7 ppm peptide mass tolerance , search engine rank 1 , 
 absolute xcorr threshold 0.4 ) . additionally , we considered only psms 
 for which the injection time did not max out ( < 500 ms ) , that is , 
 the target number of ions was reached .
note that this precaution was 
 taken to exclude the number of ions as a variable that might impair 
 the quality of fragmentation .
we calculated the average precursor 
 ion purity ( pip ) for each data set and 
 found similar values , which were approximately 95% for all three techniques . 
 together
, these stringent criteria ensure that the activation technique 
 is the only variable that controls the fragmentation behavior .
a summary 
 of the data from this direct comparison is given in table 1 .
we found that ethcd provided 248 psms 
 while these numbers were 237 and 216 for hcd and etd , respectively . 

out of the 30 unique synthetic phosphopeptides injected etd , hcd and 
 ethcd identified 21 , 22 and 24 , respectively .
we found the average 
 sequest xcorr being highest for ethcd ( 2.5 ) followed by hcd ( 1.9 ) 
 and etd ( 1.5 ) , which is in line with our previous results for nonmodified 
 peptides .
the sequest algorithm correctly 
 annotated the known phosphosites in 79% of etd and 78% of hcd data . 

significantly , for ethcd this was over 95% ( of all psms ) , which directly 
 reflects the higher spectral quality , due to the generation of both 
 b / y and c / z ions .
this initial data suggests that ethcd provides even 
 more extensive backbone fragmentation of phosphorylated peptides than 
 etd or hcd alone , facilitating sensitive phosphosite localization 
 with very high confidence .
it should be noted that the application 
 of a site localization algorithm would be prudent for real - life samples 
 since the true phosphorylation sites are unknown .
recently , taus et al . described phosphors , a novel 
 tool to improve 
 confident localization of phosphosites .
the software is based on validated peptide identifications provided 
 by database search engines and calculates site probabilities for each 
 potential phosphosite in the peptide sequence . for this study we used 
 a modified version of phosphors that also enables assessment of individual 
 phosphosite probabilities for ethcd fragmentation .
we analyzed each 
 data set using phosphors and found that it performs equally well for 
 all three fragmentation techniques .
of all true phosphosites , 96% 
 ( etd ) , 95% ( hcd ) and 97% ( ethcd ) were assigned a site probability > 99% , 
 which corresponds to a very high confidence in site localization ( table 1 ) .
together , these findings suggest that ethcd generates 
 ms / ms spectra that contain sufficient fragment ions for the unambiguous 
 and sensitive phosphorylation site localization .
next , we assessed the performance of ethcd for phosphosite 
 localization on a larger data set .
we used ti - imac material 
 for the enrichment of phosphopeptides from a tryptic digest of hela 
 cells and analyzed equal amounts ( corresponding to enriched phosphopeptides 
 from 100 g of protein ) by lc 
ms / ms with etd , hcd and 
 ethcd , respectively ( supplementary figure 1a , supporting information ) .
the 
 etd data was also searched with mascot because we found sequest to 
 perform poorly for doubly charged phosphopeptides .
note that other 
 search engines such as omssa or spectrummill might provide larger 
 number of identifications for etd data .
however , these algorithms are currently not compatible with ethcd 
 data and phosphors analysis within the proteome discoverer software 
 environment .
all identified psms were then filtered for < 1% fdr 
 using percolator to ensure consistency . in total
we identified 2217 
 ( etd ) , 4179 ( hcd ) and 3594 ( ethcd ) phospho - psms ( table 2 ) .
our initial analysis of a defined synthetic phosphopeptide 
 mixture demonstrated that ethcd performs at least on the same level 
 as hcd in terms of peptide identification .
however , the overall identification 
 success rate in the ti - imac data set was slightly lower 
 for ethcd compared to hcd .
the ms / ms spectra from the synthetic phosphopeptide 
 mixture were manually validated whereas the ti - imac data 
 set was computationally filtered to < 1% fdr .
the application of 
 ethcd , in comparison to etd or hcd alone , significantly increases 
 the number of fragment ions observed in the ms / ms scans . on the one 
 hand ethcd
spectra contain more sequence information , which is beneficial 
 for inferring the peptide sequence and ptm localization . on the other 
 hand
, these additional fragment ions may also match to random peptide 
 sequences , increasing their score and hampering the differentiation 
 between correct and incorrect matches .
similar to the increased 
 average score of decoy hits also the true hits are likely to provide 
 on average higher scores .
depending on whether the distance between 
 the two score distributions decreases or increases , the identification 
 success rate will be higher or lower .
since the i d success rate is 
 slightly lower for ethcd compared to hcd alone , the negative effect 
 of higher - scoring random matches might be more pronounced .
thus , higher 
 score cut - offs need to be applied in order to reach the desired fdr . 
 a standard target - decoy approach against 
 a reversed concatenated database revealed the fdr for ethcd ( 2.6% ) 
 being almost twice as high compared to hcd ( 1.4% ) , which provides 
 further evidence for this hypothesis .
as expected , ethcd provided a substantial increase in sequence 
 coverage ( 92% ) compared to hcd ( 81% ) and etd ( 83% ) .
remarkably , ethcd provided for 95% of all phosphosites a confident 
 site localization probability of > 99% . in the hcd data set we found 
 that 89% of all phosphosites were assigned with a confident site localization 
 probability > 99% , while this was only 81% for etd data set .
we 
 recalculated 
 these number for all peptides that contain > 2 residues that can 
 be 
 phosphorylated because singly phosphorylated peptides with only one 
 potential phosphorylation site could bias the results toward hcd . 

of all phosphosites from this subset of peptides 97% ( etcad ) , 93% 
 ( ethcd ) and 87% ( hcd ) , respectively , were assigned a localization 
 probability > 99% .
figure 1 shows an ms / ms spectrum 
 of a doubly phosphorylated peptide upon ethcd fragmentation .
the overall 
 sequence coverage is 89% taking b / y- and c / z - ions into account .
six 
 out of 18 amino acid bond cleavages are represented by c- and b - ions 
 ( referred to as  golden pairs  ) .
additionally , we observed 11 z / y - ion pairs , which strengthens 
 the argument that ethcd provides extensive sequence information that 
 facilitates pinpointing the correct phosphorylation site .
more than 
 95% of the phosphosites from all doubly phosphorylated peptides were 
 assigned with a site localization probability > 99% , highlighting 
 that 
 ethcd performs equally well with singly and doubly phosphorylated 
 peptides .
a known limitation of etd is its inability to cleave the 
 n  c bond n - terminal to proline .
figure 2 shows the ethcd spectrum of a singly phosphorylated 
 peptide that contains four serine residues .
the c- and z - ions derived 
 from the etd step cover only the n - terminal part of the peptide and 
 the site probability is only 50% .
the additional y - ions derived from 
 the subsequent hcd activation provide supporting sequence information 
 and cover also the two serine residues next to the prolines which 
 enables unambiguous phosphosite localization .
ethcd generates dual ion series that enable phosphorylation 
 site localization with very high confidence ( phosphors site probabilities : 
 t(3 ) , 0.0% ; s(6 ) , 100.0% ; s(9 ) , 100.0% ; t(15 ) , 0.0% ) .
this ethcd 
 spectrum of a doubly charged peptide that contains four serine residues , 
 one of which is phosphorylated .
etd does not cleave the n  c bond n - terminal to proline and the phosphorylation 
 site probability is only 50% based on c- and z - ions alone .
dual fragmentation 
 by ethcd generates complementary sequence information from c / z- and 
 b / y - ions ( sequest xcorr 4.10 ) . here
, the exact phosphosite is revealed 
 by y - ions that cover the corresponding phosphosite ( phosphors site 
 probabilitis : s(1 ) : 0.0 ; s(3 ) : 0.0 ; s(8 ) : 99.5 ; s(10 ) : 0.5 ) .
here we have evaluated the potential of 
 ethcd in improving the 
 analysis of phosphopeptides .
our data highlights the benefit of dual 
 ion series as generated by ethcd fragmentation .
we observed for a 
 defined phosphopeptide mixture average higher sequest xcorr values , 
 higher peptide sequence coverage and more confident phosphosite localization 
 in ethcd compared to etd and hcd .
this finding was confirmed when 
 we analyzed a complex phosphopeptide sample resulting from a ti - imac enrichment of peptides from a cellular lysate .
this 
 is in line with recent reports that showed that confidence in phosphorylation 
 site localization increases when multiple separately acquired ms / ms 
 spectra ( e.g. , etd / cid or msa / etd ) are combined for scoring . for this larger data
set , we observed that the identification success 
 rate was slightly lower for ethcd compared to hcd .
this can be attributed 
 to the use of conventional database search engines that are not optimized 
 for spectra that contain dual ion series .
however , the fact that both peptide sequence coverage and the percentage 
 of localized phosphosites are higher for ethcd than for hcd suggests 
 that once a peptide was identified , further analyses such as site 
 localization benefit from the more data - rich ethcd spectra . in ethcd 
 often c / b- and z / y - ion pairs are observed that increase the confidence 
 in a particular peptide backbone cleavage .
we speculate that the identification success rate of ethcd for phosphopeptides 
 can be improved by novel or optimized data analysis tools .
finally , 
 we reason that ethcd can also be beneficial and used to improve the 
 localization of other post - translational modifications such as ubiquitination , 
 glycosylation or acetylation .
 we recently introduced a novel scheme combining electron - transfer 
 and higher - energy collision dissociation ( termed ethcd ) , for improved 
 peptide ion fragmentation and identification . 
 we reasoned that phosphosite 
 localization , one of the major hurdles in high - throughput phosphoproteomics , 
 could also highly benefit from the generation of such ethcd spectra . 
 here , we systematically assessed the impact on phosphosite localization 
 utilizing ethcd in comparison to methods employing either etd or hcd , 
 respectively , using a defined synthetic phosphopeptide mixture and 
 also using a larger data set of ti4 + -imac enriched phosphopeptides 
 from a tryptic human cell line digest . 
 in combination with a modified 
 version of phosphors , we observed that in the majority of cases ethcd 
 generated richer and more confidently identified spectra , resulting 
 in superior phosphosite localization scores . 
 our data demonstrates 
 the distinctive potential of ethcd for ptm localization , also beyond 
 protein phosphorylation . 
Introduction
Experimental Section
Results and Discussion
Conclusions