electrospray ionization ( esi ) has revolutionized mass spectrometry ( ms ) by extending its applicability to large nonvolatile molecules . although covalent bonding and , thus , molecular weight information , is usually preserved in esi , retention of the noncovalent bonding of the solution structure has been demonstrated far less frequently [ 13 ] . esi / ms is widely used to screen for ligands that bind to target proteins ; a better understanding of the effect of esi on the noncovalent bonding in protein  ligand complexes would help evaluate the reliability of screening results . unlike collisionally activated dissociation during which different conformers of the same net charge can rearrange to structurally similar transition states prior to fragmentation , electron capture dissociation ( ecd )   can provide site - specific information on the structure and stability of gaseous protein ions ; key to this is the retention of noncovalent bonds during ecd [ 2 , 3 , 710 ] . experiments showed that solution structure is not necessarily preserved in the gas phase ; the highly stable protein cytochrome c ( g for global unfolding in solution  =  42    2  kj / mol at 30  c )   unfolds on a milliseconds timescale after transfer into the gas phase , whereas the fold of the moderately stable protein kix ( g  =  12    1  kj / mol at 27  c )   is preserved in the gas phase on a timescale of at least 4  s . unfolding of cytochrome c after desolvation was also observed in ion mobility spectrometry ( ims ) studies . the 7 + ions of ubiquitin , whose structure is believed to most closely resemble its native fold , have been studied extensively . in ims experiments , clemmer and coworkers observed substantial unfolding of 7 + ubiquitin ions on a timescale of 0.1  s after esi , whereas a recent study by wyttenbach and bowers reports conditions under which these ions do not unfold in 0.1  s . a previous ecd study has provided site - specific data on the gas phase structure and folding / unfolding of the 7 + ubiquitin ions , but these were probed > 40  s after desolvation and were shown to be stable gas phase structures substantially different from those in solution . here we report ecd data that reflect the initial structural changes of ubiquitin 7 + ions after transfer into the gas phase . experiments were performed on a 7  t fourier transform - ion cyclotron resonance ( ft - icr ) mass spectrometer ( bruker , vienna , austria ) . after esi ( 1.5 l / min ) of a 2  m ubiquitin ( sigma - aldrich , vienna , austria ) solution in 79:20:1 h2o / ch3oh / ch3cooh at ph 3 ( in which ubiquitin retains its native fold ) under conditions to minimize ion activation by collisions with background gas , ( m + 7h ) ions were accumulated in the first hexapole ( 0.1  s ) , selected by m / z in the quadrupole , accumulated in the second hexapole ( 0.1  s ) , trapped in the ft - icr cell , isolated , and irradiated with electrons ( 0.8  ev ) for 30  ms . this experiment was repeated with 5  s ion storage in the ft - icr cell prior to ecd , and with collisional activation at the head of the second hexapole . data analysis utilized snap2 ( bruker , vienna , austria ) and mmass   software . site - specific fragment yields were calculated as % -values relative to all ecd products , considering that backbone cleavage gives a pair of complementary c and z ions ( a , y ions were also included for normalization but not shown in the figures because of their marginal abundance totaling to < 4  % ): 100  %  =  0.5[c ]  +  0.5[z ]  +  0.5[a ]  +  0.5[y ]  +  [ reduced molecular ions and loss of small neutral species from the latter ] . in ubiquitin s native structure , only residues 7276 are not involved in noncovalent bonding ( figure  1 ) . if this fold were preserved in the gas phase , only fragments from cleavage at sites 7275 would be found in the ecd spectra because intramolecular noncovalent bonding would prevent separation of all other fragments [ 2 , 7 ] . however , ecd of the 7 + ions shows abundant c , z fragment ions from cleavage at many other sites ( figure  2 ) , indicating substantial unfolding after desolvation . specifically , region i ( residues 118 ) that includes -strands 1 and 2 and region iv ( residues 5976 ) that includes -strand 5 have unfolded in 57  % and 47  % of the ions after 0.2  s , respectively ( figure  2a ) . the extent of unfolding was calculated from integrated c , z fragment ion yields in each region versus those expected from random cleavage in a fully extended structure ( 100  % /75  =  1.33  % yield per cleavage site ) . the similarity of the values for regions i and iv suggests that they have separated from the hydrophobic core in the same unfolding event . from the data in figure  2a , regions ii and iii have unfolded in just 17  % and 5  % of the 7 + ions within 0.2  s , respectively.figure 1.intramolecular electrostatic interactions of ubiquitin ( pdb entry 1d3z ) with regions i ( residues 118 ) , ii ( 1935 ) , iii ( 3658 ) , and iv ( 59 - 76 ) ; basic ( k , r , n - terminus ) and acidic ( d , e , c - terminus ) residues in blue and red , respectively ; hydrogen bonds : dashed lines , solid black lines connect sequence neighbors , green lines : possible salt bridgesfigure 2.site-specific yields ( a ) , ( b ) of c , z fragments from ecd of ubiquitin ( m + 7h ) ions 0.2  s ( a ) and 5  s ( b ) after transfer into the gas phase and yield difference ( c ) ; dashed horizontal lines illustrate calculated yields for random fragmentation intramolecular electrostatic interactions of ubiquitin ( pdb entry 1d3z ) with regions i ( residues 118 ) , ii ( 1935 ) , iii ( 3658 ) , and iv ( 59 - 76 ) ; basic ( k , r , n - terminus ) and acidic ( d , e , c - terminus ) residues in blue and red , respectively ; hydrogen bonds : dashed lines , solid black lines connect sequence neighbors , green lines : possible salt bridges site - specific yields ( a ) , ( b ) of c , z fragments from ecd of ubiquitin ( m + 7h ) ions 0.2  s ( a ) and 5  s ( b ) after transfer into the gas phase and yield difference ( c ) ; dashed horizontal lines illustrate calculated yields for random fragmentation the site - specific differences of fragment yields ( figure  2c ) from ecd 0.2 and 5.2  s after transfer into the gas phase show a pattern very similar to that from ecd after 0.2  s ( figure  2a ) , indicating that the initial unfolding during the first 0.2  s continues during the next 5  s in the same manner . exceptions to this are the fragment yields at sites 15 , which do not increase in proportion from 0.2 to 5.2  s ( figure  2c ) , indicating that unfolding of residues 15 is largely complete after 0.2  s. after 5.2  s , 71  % , 22  % , 7  % , and 57  % of the ions have unfolded in regions i  iv , respectively , suggesting a regional order of stability of iii > ii > iv > i. collisional activation of the 7 + ions 0.1  s after esi under otherwise identical experimental conditions gave the fragmentation patterns shown in figure  3a , b. the data indicate an extent of unfolding of 49  % , 13  % , 17  % , and 41  % in regions i  iv after 0.2  s , respectively . again , the fragment yield differences from ecd 0.2 and 5.2  s after transfer into the gas phase ( figure  3c ) show a pattern very similar to that from ecd after 0.2  s , indicating that the unfolding during the first 0.2  s continues during the next 5  s , with 81  % , 22  % , 27  % , and 58  % of the ions unfolded in regions i  iv after 5.2  s , respectively.figure 3.as in figure  2 , but with collisional activation after 0.1  s ( 105  ev laboratory frame energy ) as in figure  2 , but with collisional activation after 0.1  s ( 105  ev laboratory frame energy ) apparently , the 26 hydrogen bonds of ubiquitin s -sheet ( figure  1 ) are insufficient to preserve the solution fold after desolvation : regions i and iv unfold in about half of the 7 + ions after just 0.2  s. on the other hand , region iii , which is the most heterogeneous in terms of secondary structure , is unfolded in only 5  % and 7  % of the ions after 0.2 and 5.2  s , respectively . substantial collisional activation is required to increase these fractions to 17  % and 27  % , indicating extensive stabilization . region ii that comprises the -helix is unfolded in only 20  % of the ions at 0.2 and 5.2  s without collisional activation , suggesting that ubiquitin s -helix structure is more stable than its -sheet in the absence of solvent . however , a recent study of kix showed that helix stability in the gas phase critically depends on additional stabilization by salt bridges . salt bridges as stabilizing structural motifs have also been suggested by williams and coworkers for gaseous peptide ions . the very same collisional activation that increases the fraction of 7 + ions unfolded in regions i and iii by 10  % and 20  % after 5.2  s , respectively , does not further unfold regions ii ( 22  % at 0.2 and 5.2  s ) and iv ( 57  % at 0.2 , 58  % at 5.2  s ) . for region ii , this is consistent with additional stabilization of the -helix by salt bridges , but structural changes are nevertheless apparent from the different ecd fragmentation patterns for region ii at 0 and 105  ev ( figure  2a , b and figure  3a , b ) . similar rearrangements are indicated for the far less compact region iv , but not for regions i and iii , whose ecd fragmentation patterns at 0 and 105  ev are very similar ( figure  2a , b and figure  3a , b ) . salt bridges , formed during the electrospray process or already present in solution , appear to play a dominant role in stabilizing regions of the native fold of ubiquitin after transfer into the gas phase . six salt bridges are possible in the most stable region iii that comprises 23 residues . the second most stable region ii ( 17 residues ) has five possible salt bridges . only two salt bridges are possible in the least stable regions i and iv , respectively . specifically , the n- and c - terminal -strands 1 and 5 are held together by hydrogen bonds , but no salt bridges can provide additional stabilization . regions i and iv comprise 36 out of ubiquitin s 76 residues ; their unfolding in just 0.2  s should result in a substantial increase in collision cross section for about half of the 7 + ions , consistent with ims data that indicate unfolding of  compact  ions ( 1000   ) to form 50  %  partially folded . however , this study also finds 15  %  elongated  structures ( > 1500   ) after 0.2  s , increasing to 25  % in 5  s. the proportion of elongated structures indicated by our ecd data is smaller , with only 7  % of the 7 + ions unfolded in region iii after 5  s. the esi and ion transmission conditions of these ims experiments appear to result in more extensive unfolding than those used here . although the timescale of unfolding of ubiquitin after transfer into the gas phase evidently depends on experimental conditions [ 17 , 18 ] , the order of unfolding established here ( region i > iv > ii > iii ) is dictated by the extent to which the loss of hydrophobic bonding is compensated by electrostatic interactions , in particular salt bridges , in different regions of the native fold . stabilization by salt bridges is also indicated for the 7 + ions probed 40  s after desolvation , which gave few fragments from cleavage at sites 1158 , consistent with disintegration of the native fold and folding into stable gas phase structures [ 15 , 17 , 22 ] . the ecd data presented here complement ims data , and allow for a structural interpretation of the unfolding of ubiquitin 7 + ions after transfer into the gas phase . solvent removal destabilizes the native fold , with regions i and iv unfolding markedly faster than regions ii and iii . we attribute the observed regional stabilization in large part to salt bridges , consistent with recent findings for the three - helix bundle protein kix . the extent to which electrostatic interactions and , in particular , salt bridges can compensate for the loss of hydrophobic bonding largely defines the timescale available for structural probing of a solution fold in the gas phase .