protein sequencing by tandem mass spectrometry ( ms / ms ) has evolved into a standard and widely applied method [ 1 , 2 ] . the most common approach in ms - based proteomics involves digestion with specific enzymes after performing a reduction / alkylation step , referred to as bottom - up or peptide - centric readout [ 35 ] .
the digestion step is often not applied for the analysis of  native  or endogenous peptides , and is even avoided for top - down protein ms [ 4 , 610 ] .
the analysis of  intact  peptides or proteins allows the characterization of one of the most common post - translational modifications ( ptms ) in proteomics , namely disulfide bonds .
s  s bridges  , stabilize the three - dimensional structure of a protein and , therefore , are crucial elements for their biological function . on the other hand , disulfide bonds can also hinder ms / ms - fragmentation in different ways . in an ms
/ ms - collision induced dissociation ( cid ) experiment primarily peptide backbone fragmentations are observed , with a preference for cleavages of the  weakest  amide bond , resulting in either b- or y - ions . in the presence of a disulfide bond , such backbone characterization is often hampered and thus poses a challenge for middle- and top - down analysis of intact proteins .
also , proper assignment becomes difficult when the fragments themselves remain connected via an s  s bridge . with the introduction of electron capture dissociation ( ecd ) and later electron transfer dissociation ( etd ) , new possibilities emerged for fragmenting disulfide bonds [ 13 , 14 ] .
s bonds , thus allowing for sequencing of regions that are involved in intra- or intermolecular linkages . nevertheless , even after cleavage of the s  s bond , the resulting  fragment ion  may still be intact because of the remaining connectivity in the peptide backbone . consequently , off - line reduction of disulfide bonds with a reagent such as dithiothreitol ( dtt ) still is a pivotal step in obtaining full sequence coverage of a protein , similar to the one carried out in a bottom - up experiment .
however , the use of dtt requires removal before ms analysis , and alternative thiol - free reducing agents also involve laborious sample preparation . with the current rise of ultrahigh resolution instruments ,
often , top - down experiments are performed using electrospray ionization ( esi ) of intact proteins ; however , there is also growing interest for in source decay ( isd ) during matrix - assisted laser desorption ionization ( maldi ) [ 17 , 18 ] .
low - ppm mass measurement precision allows a detailed analysis of for instance ptms , or even all proteoforms [ 10 , 19 ] . in this context ,
recently , it has been shown by the group of chen that electrochemical reduction of s  s bridges can be performed on - line using desorption electrospray ionization ( desi ) [ 2023 ] . in this study , we coupled electrochemistry ( ec ) to direct infusion esi - ms / ms performed on an ultrahigh resolution fourier transform ion cyclotron ( fticr ) system equipped with a 15 tesla magnet . on - line
the development of special electrodes and electronic systems has opened ways for electrochemical reactions on biomolecules , such as peptides and proteins [ 24 , 25 ] . with regard to studies on the oxidative metabolism of drugs ,
furthermore , ec can be used to reduce disulfide bonds in proteins for analytical purposes , as was recently demonstrated for -lactoglobulin a and lysozyme .
obviously , the absence of reactants is a major advantage of ec , as well as is the short time scale of interaction with the electrodes . in order to evaluate the performance of such an on - line ec - ms / ms approach , cid- and etd - spectra of two peptide model systems
it has been shown that through alternating or consecutive use of cid and etd in bottom - up proteomics lc - ms / ms experiments , based on the decision tree approach , distinct peptide sets are obtained and that the number of protein identifications ( ids ) in complex proteomes can be increased [ 25 , 2932 ] . an increased peptide sequence has also been reported from simultaneous use of two different fragmentation techniques .
similarly , the use of both fragmentation techniques provides complementary data for middle- and top - down analysis of peptides or proteins .
first , the benefits of ec on a relatively simple peptide containing only one disulfide bridge are evaluated .
oxytocin is a small hormone and neurotransmitter with an s  s bridge between the cysteine residues at positions 1 and 6 , thus fixing secondary and tertiary structure .
then , on - line ec is evaluated for a more complex middle - down peptide containing four disulfide bridges , namely hepcidin , which is a 25 amino acid - containing hormone that regulates iron metabolism and has a tightly folded hairpin structure .
oxytocin and hepcidin were purchased from sigma - aldrich ( steinheim , germany ) and anaspec ( san diego , ca , usa ) , respectively .
hplc - grade methanol , acetonitrile , isopropanol , and formic acid ( 95  % , purity ) were supplied by sigma - aldrich .
direct infusion esi - fticr experiments were performed on a bruker 15 tesla solarix fticr mass spectrometer as previously reported [ 11 , 35 ] .
briefly , a quadrupole ( q ) was used for precursor ion selection and a hexapole collision cell for cid and etd .
direct infusion esi experiments were carried out at an infusion rate of 50 l / min .
the ion funnels operated at 100 and 6.0 v , respectively , with the skimmers at 15 and 5 v. the trapping potentials were set at 1.0 and 1.0 v , the analyzer entrance was maintained at 10 v , and side kick technology was used to further optimize peak shape and signal intensity .
the required excitation power was 19  % with a pulse time of 10 s .
ms / ms - experiments were performed by cid and fragment ion mass analysis in the icr cell . for these experiments ,
the collision energy , the accumulation time in the hexapole collision cell , and the isolation window in the q were optimized for each precursor ion .
collision energies varied from 4.0 to 16.5 v , whereas the accumulation times varied from 0.5 to 10 s. both the etd reagent accumulation time and the reaction time were 0.4 s. electrochemical reduction of disulfide bond(s ) was performed and evaluated on the nine amino acid - containing peptide oxytocin ( one disulfide bridge ) and the 25 amino acid - containing peptide hepcidin ( four disulfide bridges ) .
an electrochemical cell with titanium - based electrodes ( -prepcell , cell volume 11 l ; antec , zoeterwoude , the netherlands ) , controlled by a potentiostat ( roxy potentiostat , antec ) , was used on - line in a direct infusion electrospray ionization ( di - esi ) setup on the fticr - ms system .
typically , 13 m solutions of the peptide or protein were infused through the prepcell at a flow rate of 50 l / min .
optimized settings ( i.e. , times ) were used according to manufacturer guidelines ( unpublished data ) , namely a pulse setting with a 1990 ms interval for e1 , followed by a 1010 ms interval for e2 .
oxytocin and hepcidin were purchased from sigma - aldrich ( steinheim , germany ) and anaspec ( san diego , ca , usa ) , respectively .
hplc - grade methanol , acetonitrile , isopropanol , and formic acid ( 95  % , purity ) were supplied by sigma - aldrich .
direct infusion esi - fticr experiments were performed on a bruker 15 tesla solarix fticr mass spectrometer as previously reported [ 11 , 35 ] .
briefly , a quadrupole ( q ) was used for precursor ion selection and a hexapole collision cell for cid and etd .
direct infusion esi experiments were carried out at an infusion rate of 50 l / min .
the ion funnels operated at 100 and 6.0 v , respectively , with the skimmers at 15 and 5 v. the trapping potentials were set at 1.0 and 1.0 v , the analyzer entrance was maintained at 10 v , and side kick technology was used to further optimize peak shape and signal intensity .
the required excitation power was 19  % with a pulse time of 10 s .
ms / ms - experiments were performed by cid and fragment ion mass analysis in the icr cell . for these experiments ,
the collision energy , the accumulation time in the hexapole collision cell , and the isolation window in the q were optimized for each precursor ion .
collision energies varied from 4.0 to 16.5 v , whereas the accumulation times varied from 0.5 to 10 s. both the etd reagent accumulation time and the reaction time were 0.4 s.
electrochemical reduction of disulfide bond(s ) was performed and evaluated on the nine amino acid - containing peptide oxytocin ( one disulfide bridge ) and the 25 amino acid - containing peptide hepcidin ( four disulfide bridges ) . an electrochemical cell with titanium - based electrodes (
-prepcell , cell volume 11 l ; antec , zoeterwoude , the netherlands ) , controlled by a potentiostat ( roxy potentiostat , antec ) , was used on - line in a direct infusion electrospray ionization ( di - esi ) setup on the fticr - ms system .
typically , 13 m solutions of the peptide or protein were infused through the prepcell at a flow rate of 50 l / min .
optimized settings ( i.e. , times ) were used according to manufacturer guidelines ( unpublished data ) , namely a pulse setting with a 1990 ms interval for e1 , followed by a 1010 ms interval for e2 .
oxytocin was directly infused from a syringe pump via the -prepcell into an ultrahigh resolution fticr mass spectrometer . by switching
the ec device on - and - off the disulfide bond in oxytocin either remained intact or was reduced into two sh - groups .
first , the esi solvent conditions were optimized with regard to the ms1 signal intensity and the ec conversion .
it was found that a solvent ratio of water and acetonitrile of 90/10 containing 1  % formic acid ( fa ) was optimal .
the use of methanol resulted in a decreased conversion and an increasing amount of organic solvent generally resulted in lower signal intensities .
in addition , the use of isopropanol gave unstable signal and conversion ( data not shown ) .
next , the e1 and e2 potentials were evaluated for the on - line reduction of oxytocin , using pulse settings as described in the materials and methods section .
it becomes clear that with decreasing e1 ( i.e. , from 1 to 3 v ) , the amount of  native  ( s  s bridge containing ) oxytocin decreases , and that with increasing e2 ( i.e. , from + 1 to + 2v ) the signal of reduced oxytocin is enhanced .
it was found that further increase of e2 resulted in a ( small ) decrease of reduced oxytocin . as can be seen from figure  2a , the maximum conversion of oxytocin into its reduced form was approximately 80  % at e2 = 2.2 v.figure 1schematic experimental setup for the reduction of disulfide bonds and on - line infusion into an ultrahigh resolution fticr mass spectrometer .
collision - induced dissociation ( cid ) and electron - transfer dissociation ( etd ) were used to characterize both native and reduced form of oxytocin and hepcidinfigure 2evaluation of the -prepcell potentials for the on - line reduction of the disulfide bridge of oxytocin ( a ) .
the maximum conversion rate of approximately 80  % was obtained at e1 = 2.2 v and e2 = 2.2 v. evaluation of the conversion rate ( red - ox ) monitoring the signal intensity of the doubly charged oxytocin ( b ) measured with a resolving power of 250,000 .
the two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ; left - hand panel ) and reduced oxytocin ( [ mred + 2h ] ; right - hand panel ) were resolved . the case of a partial reduction of oxytocin is shown in the middle panel . here , three peaks are resolved at m / z 505.2 shown in the enlarged part , corresponding to the third isotopic peak of [ m + 2h ] with s and c2 , respectively , and the monoisotopic peak of [ mred + 2h ] 
  schematic experimental setup for the reduction of disulfide bonds and on - line infusion into an ultrahigh resolution fticr mass spectrometer .
collision - induced dissociation ( cid ) and electron - transfer dissociation ( etd ) were used to characterize both native and reduced form of oxytocin and hepcidin evaluation of the -prepcell potentials for the on - line reduction of the disulfide bridge of oxytocin ( a ) .
the maximum conversion rate of approximately 80  % was obtained at e1 = 2.2 v and e2 = 2.2 v. evaluation of the conversion rate ( red - ox ) monitoring the signal intensity of the doubly charged oxytocin ( b ) measured with a resolving power of 250,000 .
the two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ; left - hand panel ) and reduced oxytocin ( [ mred + 2h ] ; right - hand panel ) were resolved .
the case of a partial reduction of oxytocin is shown in the middle panel . here , three peaks are resolved at m / z 505.2 shown in the enlarged part , corresponding to the third isotopic peak of [ m + 2h ] with s and c2 , respectively , and the monoisotopic peak of [ mred + 2h ] 
  note that the ultrahigh resolving power of the fticr system allowed the detailed analysis of two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ) and reduced oxytocin ( [ m + 2h + 2h ] , further referred to as ( [ mred + 2h ] ) .
the differences within the third isotopic peak at approximately m / z 505.2 are shown in figure  2b . in the case of native oxytocin ,
this signal consists of two peaks attributable to the presence or absence of a s isotope , with a difference of 5.6 milli - mass units ( left - hand panel of figure  2b ) . in the case of reduced oxytocin ,
the corresponding signal at m / z 505.2 has turned into a monoisotopic signal and , thus , contains only one peak , which is observed at 4.3 milli - mass units higher than the third isotopic peak of doubly protonated native oxytocin ( right - hand panel of figure  2b ) .
both species are present when the on - line reduction of the disulfide bond is not complete , exemplified by the overlap of two isotopic profiles ( middle panel of figure  2b ) .
subsequently , the system as depicted in figure  1 was used to generate ms / ms - data from both native and reduced oxytocin .
the cid spectrum of doubly protonated native oxytocin is depicted in figure  3 , as well as the one that was obtained after on - line reduction of the s 
the large majority of fragment ions could be explained from  standard  b / y - fragmentation , including neutral losses .
in addition , a few ions were assigned as a - ions or as internal fragments resulting from two - bond cleavages .
the fragment ions observed from native oxytocin , and the resulting sequence coverage , were in good agreement with previously obtained data from sustained off - resonance induced ( sori ) cid - experiments , with dominant cleavages of the three amino acid  tail  and minor fragmentation of the six amino acid - containing ring (  tocin ring  ) .
the only noticeable difference between the current and the sori - cid data was the absence of a b3-ion in the cid spectrum of native oxytocin , next to variation in the ion abundances . from the comparison of the cid spectra of native and reduced oxytocin ,
it becomes evident that many additional fragment ions from tocin - ring cleavages have appeared after the on - line ec reduction of the s 
s bridge ( e.g. , b2/y7 , b3/y6 , b4/y5 , b5/y4 ) . as a result
similarly , the application of etd yielded large differences between fragmentation spectra of native and reduced oxytocin .
this is depicted in figure  4 the benefits in terms of sequence coverage are not as obvious as in the case of cid spectra , albeit that the number of sequence - informative fragment ions doubled from 8 to 16 and that the total number of ( assigned ) fragment ions was higher for the reduced species . upon comparison of the etd spectrum of doubly protonated native oxytocin with previous results obtained from ecd experiments , it was noted that the type of fragments were the same , i.e. typical b / y - fragments and typical c - fragments .
interestingly , the number of tocin - ring cleavages was lower in etd than in ecd spectra : b3- , b5- , and c5-ions were absent in etd , as well as y4- , y5- and y7-ions .
on the other hand , in the etd spectra ,  new  y8- , b8- , c7- , and c8-ions were observed .
it was further noted that both b / y- and c / z - fragments were observed in the etd spectrum of ec - reduced , doubly protonated oxytocin . this is somewhat different from the previous ecd results , in which reduced and carboxymethylated doubly protonated oxytocin yielded solely b / y - fragments.figure 3cid spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin . the online reduction results in a richer ms / ms spectrum and , consequently , a more comprehensive characterization of the peptide sequencefigure 4etd spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin
. the differences between the two spectra are less apparent than in the case of cid spectra .
the on - line reduction of the disulfide bridge results in a more comprehensive characterization of the tocin - ring cid spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin .
the online reduction results in a richer ms / ms spectrum and , consequently , a more comprehensive characterization of the peptide sequence etd spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin .
the differences between the two spectra are less apparent than in the case of cid spectra .
the on - line reduction of the disulfide bridge results in a more comprehensive characterization of the tocin - ring the system depicted in figure  1 was used for a top - down analysis of the 25 amino acid - containing hormone hepcidin .
the cid spectrum of native hepcidin was recorded , showing mostly typical b / y - fragments and a few a - fragment ions . from the sequence coverage shown in figure  5 ,
it becomes clear that the observed fragment ions resulted solely from peptide backbone cleavages that are not involved in the region containing the four disulfide bonds .
in other words , the disulfide bonds clearly hamper sequencing the part from cys7 to cys23 . after on - line
electrochemical reduction of all four s  s bridges , the cid spectrum contained many more fragment ions , as can be seen in figure  5 .
the ms / ms - data are still dominated by b- and y - fragments and , in addition , more internal fragments were observed ( resulting from two - bond cleavages ) .
the beneficial effect of using on - line ec becomes clear from the sequence coverage : only seven bonds were fragmented in the native species , whereas 21 out of 24 peptide bonds were cleaved in the fully reduced hepcidin .
previous ms identifications of hepcidin were based mostly on ( accurate ) mass measurements [ 34 , 37 ] . to our knowledge , this is the first study where such high sequence coverage of hepcidin was obtained , although lc - ms / ms methods have been used to identify and quantify hepcidin in body fluids [ 38 , 39].figure 5cid spectrum of native ( a - i , -ii , -iii , -iv , and -v ) and on - line reduced ( b - i , -ii , -iii , -iv , and -v ) hepcidin .
the fragmentation of the native hepcidin led to backbone cleavages that are not involved in the region containing the four disulfide bonds .
the on - line reduction of the four s  s bridges prior the ms / ms experiment led to a more extensive fragmentation resulting in 21 out of 24 cleaved peptide bonds .
the following internal fragments were also observed as doubly charged ions : ( 1 ) cifccgcchrskcgmcck ; ( 2 ) cifccgcchrskcgmcck ; ( 3 ) picifccgcchrskcgmcck ; ( 4 ) fpicifccgcchrskcgmcck ; ( 5 ) ccgcchrskcgmcck ; ( 6 ) fccgcchrskcgmcck ; ( 7 ) cifccgcchrskcgmcck ; ( 8) picifccgcchrskcgmcck cid spectrum of native ( a - i , -ii , -iii , -iv , and -v ) and on - line reduced ( b - i , -ii , -iii , -iv , and -v ) hepcidin .
the fragmentation of the native hepcidin led to backbone cleavages that are not involved in the region containing the four disulfide bonds .
the on - line reduction of the four s  s bridges prior the ms / ms experiment led to a more extensive fragmentation resulting in 21 out of 24 cleaved peptide bonds .
the following internal fragments were also observed as doubly charged ions : ( 1 ) cifccgcchrskcgmcck ; ( 2 ) cifccgcchrskcgmcck ; ( 3 ) picifccgcchrskcgmcck ; ( 4 ) fpicifccgcchrskcgmcck ; ( 5 ) ccgcchrskcgmcck ; ( 6 ) fccgcchrskcgmcck ; ( 7 ) cifccgcchrskcgmcck ; ( 8) picifccgcchrskcgmcck in summary , the presented study here shows that disulfide bridges can be successfully reduced in an on - line esi set - up using electrochemistry .
these findings are similar to those obtained by other authors using desi [ 2022 ] .
it will be of great interest to further study the coupling of ec with lc - based separation of proteins and on - line digestion , as this might finally lead to a fully integrated workflow overcoming the reduction / alkylation step in standard bottom - up proteomics .
in addition , the coupling of ec with lc - based peptide separation may open new avenues in the field of de novo and middle - down sequencing of ( large ) peptides .
oxytocin was directly infused from a syringe pump via the -prepcell into an ultrahigh resolution fticr mass spectrometer . by switching
the ec device on - and - off the disulfide bond in oxytocin either remained intact or was reduced into two sh - groups .
first , the esi solvent conditions were optimized with regard to the ms1 signal intensity and the ec conversion .
it was found that a solvent ratio of water and acetonitrile of 90/10 containing 1  % formic acid ( fa ) was optimal .
the use of methanol resulted in a decreased conversion and an increasing amount of organic solvent generally resulted in lower signal intensities .
in addition , the use of isopropanol gave unstable signal and conversion ( data not shown ) .
next , the e1 and e2 potentials were evaluated for the on - line reduction of oxytocin , using pulse settings as described in the materials and methods section .
it becomes clear that with decreasing e1 ( i.e. , from 1 to 3 v ) , the amount of  native  ( s  s bridge containing ) oxytocin decreases , and that with increasing e2 ( i.e. , from + 1 to + 2v ) the signal of reduced oxytocin is enhanced .
it was found that further increase of e2 resulted in a ( small ) decrease of reduced oxytocin . as can be seen from figure  2a , the maximum conversion of oxytocin into its reduced form was approximately 80  % at e2 = 2.2 v.figure 1schematic experimental setup for the reduction of disulfide bonds and on - line infusion into an ultrahigh resolution fticr mass spectrometer .
collision - induced dissociation ( cid ) and electron - transfer dissociation ( etd ) were used to characterize both native and reduced form of oxytocin and hepcidinfigure 2evaluation of the -prepcell potentials for the on - line reduction of the disulfide bridge of oxytocin ( a ) .
the maximum conversion rate of approximately 80  % was obtained at e1 = 2.2 v and e2 = 2.2 v. evaluation of the conversion rate ( red - ox ) monitoring the signal intensity of the doubly charged oxytocin ( b ) measured with a resolving power of 250,000 .
the two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ; left - hand panel ) and reduced oxytocin ( [ mred + 2h ] ; right - hand panel ) were resolved . the case of a partial reduction of oxytocin is shown in the middle panel . here , three peaks are resolved at m / z 505.2 shown in the enlarged part , corresponding to the third isotopic peak of [ m + 2h ] with s and c2 , respectively , and the monoisotopic peak of [ mred + 2h ] 
  schematic experimental setup for the reduction of disulfide bonds and on - line infusion into an ultrahigh resolution fticr mass spectrometer .
collision - induced dissociation ( cid ) and electron - transfer dissociation ( etd ) were used to characterize both native and reduced form of oxytocin and hepcidin evaluation of the -prepcell potentials for the on - line reduction of the disulfide bridge of oxytocin ( a ) .
the maximum conversion rate of approximately 80  % was obtained at e1 = 2.2 v and e2 = 2.2 v. evaluation of the conversion rate ( red - ox ) monitoring the signal intensity of the doubly charged oxytocin ( b ) measured with a resolving power of 250,000 .
the two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ; left - hand panel ) and reduced oxytocin ( [ mred + 2h ] ; right - hand panel ) were resolved .
the case of a partial reduction of oxytocin is shown in the middle panel . here , three peaks are resolved at m / z 505.2 shown in the enlarged part , corresponding to the third isotopic peak of [ m + 2h ] with s and c2 , respectively , and the monoisotopic peak of [ mred + 2h ] 
  note that the ultrahigh resolving power of the fticr system allowed the detailed analysis of two overlapping isotopic profiles of the doubly protonated molecular ions of native ( [ m + 2h ] ) and reduced oxytocin ( [ m + 2h + 2h ] , further referred to as ( [ mred + 2h ] ) .
the differences within the third isotopic peak at approximately m / z 505.2 are shown in figure  2b . in the case of native oxytocin ,
this signal consists of two peaks attributable to the presence or absence of a s isotope , with a difference of 5.6 milli - mass units ( left - hand panel of figure  2b ) . in the case of reduced oxytocin ,
the corresponding signal at m / z 505.2 has turned into a monoisotopic signal and , thus , contains only one peak , which is observed at 4.3 milli - mass units higher than the third isotopic peak of doubly protonated native oxytocin ( right - hand panel of figure  2b ) .
both species are present when the on - line reduction of the disulfide bond is not complete , exemplified by the overlap of two isotopic profiles ( middle panel of figure  2b ) .
subsequently , the system as depicted in figure  1 was used to generate ms / ms - data from both native and reduced oxytocin .
the cid spectrum of doubly protonated native oxytocin is depicted in figure  3 , as well as the one that was obtained after on - line reduction of the s 
the large majority of fragment ions could be explained from  standard  b / y - fragmentation , including neutral losses .
in addition , a few ions were assigned as a - ions or as internal fragments resulting from two - bond cleavages .
the fragment ions observed from native oxytocin , and the resulting sequence coverage , were in good agreement with previously obtained data from sustained off - resonance induced ( sori ) cid - experiments , with dominant cleavages of the three amino acid  tail  and minor fragmentation of the six amino acid - containing ring (  tocin ring  ) .
the only noticeable difference between the current and the sori - cid data was the absence of a b3-ion in the cid spectrum of native oxytocin , next to variation in the ion abundances . from the comparison of the cid spectra of native and reduced oxytocin ,
it becomes evident that many additional fragment ions from tocin - ring cleavages have appeared after the on - line ec reduction of the s 
s bridge ( e.g. , b2/y7 , b3/y6 , b4/y5 , b5/y4 ) . as a result
similarly , the application of etd yielded large differences between fragmentation spectra of native and reduced oxytocin .
this is depicted in figure  4 the benefits in terms of sequence coverage are not as obvious as in the case of cid spectra , albeit that the number of sequence - informative fragment ions doubled from 8 to 16 and that the total number of ( assigned ) fragment ions was higher for the reduced species . upon comparison of the etd spectrum of doubly protonated native oxytocin with previous results obtained from ecd experiments , it was noted that the type of fragments were the same , i.e. typical b / y - fragments and typical c - fragments .
interestingly , the number of tocin - ring cleavages was lower in etd than in ecd spectra : b3- , b5- , and c5-ions were absent in etd , as well as y4- , y5- and y7-ions .
on the other hand , in the etd spectra ,  new  y8- , b8- , c7- , and c8-ions were observed .
it was further noted that both b / y- and c / z - fragments were observed in the etd spectrum of ec - reduced , doubly protonated oxytocin . this is somewhat different from the previous ecd results , in which reduced and carboxymethylated doubly protonated oxytocin yielded solely b / y - fragments.figure 3cid spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin . the online reduction results in a richer ms / ms spectrum and , consequently , a more comprehensive characterization of the peptide sequencefigure 4etd spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin
. the differences between the two spectra are less apparent than in the case of cid spectra .
the on - line reduction of the disulfide bridge results in a more comprehensive characterization of the tocin - ring cid spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin .
the online reduction results in a richer ms / ms spectrum and , consequently , a more comprehensive characterization of the peptide sequence etd spectrum of doubly protonated native ( a ) and on - line reduced ( b ) oxytocin .
the differences between the two spectra are less apparent than in the case of cid spectra .
the on - line reduction of the disulfide bridge results in a more comprehensive characterization of the tocin - ring
the system depicted in figure  1 was used for a top - down analysis of the 25 amino acid - containing hormone hepcidin .
the cid spectrum of native hepcidin was recorded , showing mostly typical b / y - fragments and a few a - fragment ions . from the sequence coverage shown in figure  5 ,
it becomes clear that the observed fragment ions resulted solely from peptide backbone cleavages that are not involved in the region containing the four disulfide bonds .
in other words , the disulfide bonds clearly hamper sequencing the part from cys7 to cys23 . after on - line
electrochemical reduction of all four s  s bridges , the cid spectrum contained many more fragment ions , as can be seen in figure  5 .
the ms / ms - data are still dominated by b- and y - fragments and , in addition , more internal fragments were observed ( resulting from two - bond cleavages ) .
the beneficial effect of using on - line ec becomes clear from the sequence coverage : only seven bonds were fragmented in the native species , whereas 21 out of 24 peptide bonds were cleaved in the fully reduced hepcidin .
previous ms identifications of hepcidin were based mostly on ( accurate ) mass measurements [ 34 , 37 ] . to our knowledge , this is the first study where such high sequence coverage of hepcidin was obtained , although lc - ms / ms methods have been used to identify and quantify hepcidin in body fluids [ 38 , 39].figure 5cid spectrum of native ( a - i , -ii , -iii , -iv , and -v ) and on - line reduced ( b - i , -ii , -iii , -iv , and -v ) hepcidin .
the fragmentation of the native hepcidin led to backbone cleavages that are not involved in the region containing the four disulfide bonds .
the on - line reduction of the four s  s bridges prior the ms / ms experiment led to a more extensive fragmentation resulting in 21 out of 24 cleaved peptide bonds .
the following internal fragments were also observed as doubly charged ions : ( 1 ) cifccgcchrskcgmcck ; ( 2 ) cifccgcchrskcgmcck ; ( 3 ) picifccgcchrskcgmcck ; ( 4 ) fpicifccgcchrskcgmcck ; ( 5 ) ccgcchrskcgmcck ; ( 6 ) fccgcchrskcgmcck ; ( 7 ) cifccgcchrskcgmcck ; ( 8) picifccgcchrskcgmcck cid spectrum of native ( a - i , -ii , -iii , -iv , and -v ) and on - line reduced ( b - i , -ii , -iii , -iv , and -v ) hepcidin . the fragmentation of the native hepcidin led to backbone cleavages that are not involved in the region containing the four disulfide bonds .
the on - line reduction of the four s  s bridges prior the ms / ms experiment led to a more extensive fragmentation resulting in 21 out of 24 cleaved peptide bonds . the following internal fragments were also observed as doubly charged ions : ( 1 ) cifccgcchrskcgmcck ; ( 2 ) cifccgcchrskcgmcck ; ( 3 ) picifccgcchrskcgmcck ; ( 4 ) fpicifccgcchrskcgmcck ; ( 5 ) ccgcchrskcgmcck ; ( 6 ) fccgcchrskcgmcck ; ( 7 ) cifccgcchrskcgmcck ; ( 8) picifccgcchrskcgmcck in summary , the presented study here shows that disulfide bridges can be successfully reduced in an on - line esi set - up using electrochemistry .
these findings are similar to those obtained by other authors using desi [ 2022 ] .
it will be of great interest to further study the coupling of ec with lc - based separation of proteins and on - line digestion , as this might finally lead to a fully integrated workflow overcoming the reduction / alkylation step in standard bottom - up proteomics .
in addition , the coupling of ec with lc - based peptide separation may open new avenues in the field of de novo and middle - down sequencing of ( large ) peptides .
 particularly in the field of middle- and top - down peptide and protein analysis , disulfide bridges can severely hinder fragmentation and thus impede sequence analysis ( coverage ) . here 
 we present an on - line / electrochemistry / esi - fticr - ms approach , which was applied to the analysis of the primary structure of oxytocin , containing one disulfide bridge , and of hepcidin , containing four disulfide bridges . 
 the presented workflow provided up to 80  % ( on - line ) conversion of disulfide bonds in both peptides . with minimal sample preparation , 
 such reduction resulted in a higher number of peptide backbone cleavages upon cid or etd fragmentation , and thus yielded improved sequence coverage . 
 the cycle times , including electrode recovery , were rapid and , therefore , might very well be coupled with liquid chromatography for protein or peptide separation , which has great potential for high - throughput analysis . 
Introduction
Materials and Methods
Chemicals
Mass Spectrometry
Electrochemical Reduction
Results and Discussion
On-line EC-MS/MS of Oxytocin
On-line EC-MS/MS of Hepcidin