3. Investigation of the Mechanisms of Inter- and Intramolecular Disulfide Bond Cleavages
4.3. Results and Discussion
4.3.1. Rationale of CIT Design
At the inception of this study, a key goal was to find an appropriate gas-phase fragmentation pathway for the formation of the reporter ions. At that time, we were interested in the application of bio-orthogonal azide-alkyne “click” cycloaddition reactions218-220 to MS-based proteomics studies. Observation of a highly selective gas-phase fragmentation triggered by a nucleophilic substitution of N3 nitrogen in the 1,2,3-triazole ring competitive with the formation of b- and y-
type ions in CID of covalently labeled peptides inspired us to create novel isobaric tags (Scheme 3).
Figure 4.2 depicts the structure of CIT and the construction of the theoretical N-plex reagents.
CIT is composed of three parts: the reporter ion group, the mass balance group and the amine reactive group found in other commercially available isobaric tags.
The major improvement of CIT that distinguishes it from other isobaric tags is the modularization of the isotope-coded residues, both for the reporter ion group and the mass balance group. Any groups (R) that do not contain other reactive or interfering functionalities can be implemented into the current CIT design by inserting a good leaving group such as bromine, iodine or tosylate. Via a simple SN2 reaction in the mild conditions employing DMF/NaN3, the isotope-coded reporter ion group can be easily prepared from an activated R group (Figure 4.2).
Each isobaric pair of Rm−X (X: leaving group) and Rn−N3 forms a building block for an isobaric tag with a certain reporter ion mass (Rn + 123 Da). By preparing a set of the N different isotope- coded R−X, it is possible to construct the N-plex isobaric reagents. This modularity of CIT significantly reduces the effort and cost of synthesis. This feature is made possible by the newly discovered low energy gas phase fragmentation pathway depicted in Scheme 4.3, which occurs regardless of the attached R groups. In addition, the mass of the reporter ion is tunable; this property enables us to bypass the mass cut-off problem in ion trap mass spectrometers and target open windows of m/z values normally found in peptide tandem MS (e.g., sequence ions, immonium ions or internal fragments).
Figure 4.2 Design and structure of CIT
N N
N MS/MS
N N N
N O
O Rm
O O
Rn Peptide Labeling
Reporter Ion Group Mass Balance Group
Amine Reactive Group
Rn+ Rm= constant
Rn
Rm X NaN3 Rn N3
Click reaction Alkylation
R1 X RN N3 Rm X Rn N3 RN X R1 N3 N-plex
Isobaric Reagents
Alkylation
Reagent Click Reaction
Reagent Reporter Ion N N N
Rn
(mass = Rn+ 123) X: leaving group
Duplex embodiment in this report Br
CD2 Br D
D D
N3 CD2
N3 D
D D
N N N
N N N D2C R1:
R2: D
D D m/z 164 m/z 169 Light Tag Heavy Tag
a)
b)
c)
a) The components of N-plex CIT reagents: the reporter ion group, the mass balance group and the amine reactive group. b) Each reporter ion group and mass balance group can be prepared from a series of isotope-coded iodinated Rn groups. CIT labeled peptides are fragmented by various ion activation methods (e.g., PQD, beam-type CID, and High energy Collisional Dissociation (HCD)), yielding the reporter ions whose masses are Rn + 123 Da. c) the duplex embodiment of the CIT reagents in this report by using allyl bromide-d0 and d5. Note that the reporter ion is formed regardless of the structure of the attached Rn or Rm groups.
These new discoveries are applied to the creation of a prototype CIT using allyl bromide-d0 and d5 as the isotope-coded starting materials. Duplex CIT reagents were synthesized, with heavy and light isotopes having the reporter ions at m/z 164 and 169, respectively. The size of the overall modification by this duplex CIT reagent is 279 Da, which is not much larger than most of the commercially available isobaric tags (iTRAQ 4-plex, 144 Da; TMT 6-plex, 224 Da; iTRAQ 8- plex, 304 Da). Only effective collision between peptide amine and N-hydroxysuccinimide (NHS) of CIT induces the actual coupling reaction. Therefore, if the size of a certain isobaric tag is relatively large, the kinetics of conjugation can be adversely affected due to decreased frequency of the effective collision. Because the size of CIT is comparable to other isobaric tags, no significant slowdown in CIT labeling is expected.
We adopt the NHS group for facile amine-reactive coupling to peptides as in other commercially available isobaric tags. NHS has been popular in bioconjugation due to the compatibility with most biological buffer solutions. Most importantly, its target functional groups (N-termini of peptides and the ε-amine of lysine) are ubiquitous among tryptic peptides.
The reporter ion fragmentation pathway of CIT is proposed to occur as depicted in Scheme 4.3.
In multiply protonated CIT-labeled peptides, the tertiary amine in the CIT reagent would be protonated due to its higher proton affinity than most backbone amides and amino acid side- chains. A nucleophilic attack of N3 of the 1,2,3-triazole ring to the Cα of the protonated N,N- alkylated alanine residue in the CIT reagent releases a stable quaternary ammonium reporter ion, forming a six-membered ring.
The energetics of reporter ion formation is investigated by density functional theory calculations (Figure 4.3). If this process is significantly favored compared to backbone fragmentation, less sequence information would be acquired by having fewer and weaker intensity b- and y-type ions in the MS/MS spectrum. It is desirable that activation parameters
T fo B ac
The model sy or calculation B3LYP/6-311+
ctivation for a
Fi
ystem, N,N-di n. Geometry ++G(d,p) lev amide cleavag
igure 4.3 Ene
imethyl-4-(1- y optimizatio vel of theory ge to form b-
ergetics of rep
-methyl-1H-1 on, thermoch . The shaded
and y-type io
porter ion form
1,2,3-triazol-4 hemical calcu d area indica ons via collisi
mation
4-yl)butan-1-a ulation were ates the rang
ional activatio
amine was c performed e of enthalpi on.
hosen using ies of
associated with reporter ion formation are balanced with those of backbone fragmentation. This ensures that accurate protein quantification is achieved while not reducing sequencing efficacy using MS/MS. In our calculation model, the formation of the reporter ion is simulated by the N- protonated N,N-dimethyl-4-(1-methyl-1H-1,2,3-triazol-4-yl)butan-1-amine. At the B3LYP/6- 311++G(d,p) level of theory, the reaction barrier and enthalpy at 1 atm and 298.15 K are determined as 33.7 and 13.3 kcal/mol, respectively (Figure 4.3). The usual reaction barrier for the amide bond cleavage is ranged from 25 to 40 kcal/mol.161 Therefore, it is expected that most of the backbone cleavages occur as efficiently as the formation of the reporter ion.