[proteomics] Evaluation of Low Energy CID and ECD Fragmentation Behavior of Mono-Oxidized Thio-Ether Bonds in Peptides

  • From: Mavi Gozler <mavigozler@xxxxxxxxx>
  • To: proteomics@xxxxxxxxxxxxx
  • Date: Sat, 16 Dec 2006 03:03:42 -0800 (PST)


The article below talks about how thio-ethers that result from modification of 
Cys in peptides using different alkylating agents (iodoacetamide, 
maleimide-type reagents, 4-vinylpyridine) can become oxidized (i.e.  
-CH2-S-CH2- becomes -CH2-S(=O)-CH2-) and this makes the sulfoxyl bond 
subsceptible on either side to breaking under the conditions of low energy 
collisions in the mass spectrometer.  The final figure provides the 
hypothetical mechanism by which elimination occurs on either side of the sulfur 
atom to produce the alkylating agent (R) and immediate loss of water (H2O) with 
formation of an unstable sulfenic acid:

   aa-CH2-S(=O)-R(-H)  -->  aa-CH2-S-OH + R
  aa-CH2-S-OH         -->  aa-CH=S + H2O

 where aa is the amino acid backbone).  The break on the other side is 

   aa(-H)-CH2-S(=O)-R  -->  aa=CH2  + HO-S-R
to form a dehydroalanine.  Both collision-induced dissocation (CID) and 
electron capture dissociation (ECD) were the means of studying the 
fragmentation spectra in an FTICR-MS.

 Chowdhury SM, Munske GR, Ronald RC, Bruce JE   (2007)   J. Am. Soc. Mass 
Spectrom.   18,   xxx           

 Evaluation of Low Energy CID and ECD Fragmentation Behavior of Mono-Oxidized 
Thio-Ether Bonds in Peptides 

Bottom-up (expression) proteomics involves protein isolation and comparison of 
its tryptic peptide masses obtained by MS to a database set of sequences for 
high confidence identifications.  In the process, proteins must be reduced to 
break -S-S- bonds whose reformation is blocked with an alkylating agent in a 
standard nucleophilic substitution, with other reagents being Michael 
acceptors.  The product has a thioether bond (-R-S-R&#8242;-).  If 
iodoacetamide was used, a neutral fragment is lost during low energy MS/MS with 
formation of dehydroalanine (CH2=C(NHR)-COR&#8242;) at the residue.  The part 
of the side chain lost is a sulfenic acid (RSOH, where R is the rest of the 
structure depending on blocking agent used). 

For this study, two C-containing peptides (SEVAHRFKC [Pep1] and CHWKQNDEQM 
[Pep2]) were synthesized on ABi 431-A, one with N-terminal and other with 
C-terminal cysteines.  Pep1 was treated with iodoacetamide then with 3% H2O2 15 
min, desalted with C18 ZipTip, vacuum dried and injected to ESI-FTICR MS 
(Bruker Daltonics 7T APEX Q-FTICR, with CID spectra calibrated using substance 
P, bradykinin, and Pep1) in 49% MeOH/2% acetic acid solvent with a 20 µl/h 
nanospray.  A [M+2H]2+ ion at m/z 575.2794 representing oxidized alkylated 
peptide was passed as a parent to the collision cell with the trap voltage 
altered to -1V, -6V, -9V, and -12V.  At -6V (low energy collision) a peak at 
m/z 521.7770 shows at high intensity with predicted neutral loss of RSOH (where 
R=58.0292).  Accurate mass calculation shows this neutral fragment mass (RSOH, 
C2H5NO2S) to be 107.0042 with the observed(measured) being 107.0048, an error 
of 5 ppm.  At -9V the ion also is at high intensity, thus at an
 energy low enough that no other fragments but the neutral loss of RSOH 
occurring. At -12V, the relative intensity was even greater for the ion. 

Besides iodoacetamide, other blocking agents were studied. Maleimide modifies 
thiols through Michael addition at a reactive alkenyl bond. 3-maleimidpropionic 
acid was used to modify Pep1.  A [M+2H]2+ ion at m/z 631.2848 is observed and 
collisional trap voltage altered. Ions at m/z 521.7758 and 537.7613 appear, the 
latter its intensity increasing with collision voltage, while the former had a 
low intensity that did not change with increasing voltage.  Thus there is a 
loss of R + H2 from oxidized peptide, whose calculated mass is 187.0480 and 
whose measured mass is 187.0470. 

4-vinylpyridine is another Cys-modifying agent.  It differs from being 
maleimide-type in that there is asymmetry in the alkenyl bond. Presence of an 
ion at m/z 599.2939 shows the [M+2H]2+ vinylpyridine-modified Pep1.  With 
increasing collisional trap voltage, an ion at m/z 537.7590 increasing relative 
to base peak with increasing voltage indicates neutral loss fragment R + water. 
 Also increasing in relative intensity with collisional voltage increase is an 
ion at m/z 1074.5105 (appearing strong in -9V spectrum, 1074.5125 in -12V 
spectrum), representing a +1 peptide with R + H2 loss. Also present to a small 
degree was the m/z 512.7590 ion, showing a RSOH loss. 

The N-terminal Cys peptide Pep2 was also used to see if it had the same effect. 
 Peptide mass was checked for modified (and unoxidized) peptide. In addition an 
electron capture dissociation (ECD) was used to generate fragments (features a 
hot hollow cathode brought to 1.8-1.9 amperes and located outside ICR cell, 
with side kick trapping voltage from +6V to -6V, and electrons accelerated with 
3 eV and 200 ms electron injection time). Because Pep2 also has Met, it gets 
oxidized with the peroxide treatment. The 3-maleimidopropionic-modified and 
oxidized peptide shows [M+2H]2+ ion at m/z 760.2837.  Increasing collisional 
voltage produces ion at m/z 666.7595 representing 2+ ion with R + H2O neutral 

The presence of Met as a sulfoxide is not different than an oxidized thio-ether 
bond off the Cys side chain.  -15V spectra were obtained on oxidized and 
untreated peptides and showed similar fragmentation patterns, indicating that 
the Met sulfoxides do not fragment in the same way as oxidized thio-ether 
modifications of Cys.  ECD spectra of oxidized iodoacetamide-treated Pep1 
generated three c ions, c6, c7, and c8 (SEVAHR.F.K.C, where c6 is SEVAHR, 
etc.).  Also present is an [M+H]+ ion at m/z 1149.57 and 1091.57, the latter 
representing loss of iodoacetamide from modified and oxidized peptide.  Thus 
R+H2 and RSOH losses are seen, but at energies that also cause peptide 
fragmentation. Fragmentation patterns using ECD were similar for 
maleimide-treated oxidized Pep2 and iodoacetamide-treated oxidized Pep1.  
Generally with ECD based spectra there is more fragmentation of the peptide 
backbone, and in iodoacetamide-treated there is RSOH and R + H2O neutral 
losses, but with
 maleimide treatment on R + H2O losses are seen. 

The mechanism of gas phase &#946; elimination of oxidized thio-ethers involves 
a coordination of an acidic (highly positively polarized) hydrogen (&#946; to 
the sulfur atom and/or carbonyl group) to a non-bonding electron pair on the 
oxygen atom forming the oxidized thio-ether.  Arrangements of the bonds breaks 
the thio-ether bond to the R group, leaving -CH2-S-OH sulfenic acid on the side 
and restoration of the R group.  The sulfenic acid group is highly unstable, 
and leads to water loss to produce -CH=S sulfide (relatively more stable).  
This accounts for the R + H2O loss. 

In the other mechanism, the other thio-ether bond oriented to the side chain 
amino acid is broken (rather than the one connecting the R group modifier).  
This is because the negative polar oxygen atom instead bonds to the H atom on 
the &#945;-carbon of the peptide backbone, with a concerted breaking of the 
-C-CH2-S(=O)- thio-ether bond to produce dehydroalalanine and the RSOH leaving 
group.  The modifying group will dictate which mechanism dominates, if one 
dominates at all.


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