A systematic study was carried out to examine the effects of acidic amino acid residues and the position of the acidic group on the cyclization of b ions. The study utilized the model C-terminal amidated peptides XAAAAAA, AXAAAAA, AAXAAAA, AAAXAAA, AAAAXAA, AAAAAXA, AAAAAAX, XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX, where X is a glutamic acid (E) or aspartic acid (D) residue. The CID mass spectra of b n (where n=7 and 8) ions derived from XAAAAAA, AAAXAAA, AAAAAAX and XXAAAAAA, AAXXAAAA, AAAAXXAA, and AAAAAAXX exhibited very similar fragmentation patterns for both the glutamic and the aspartic acid peptide series. The CID mass spectra of MH + derived from model peptides presented substantial direct and non-direct sequence b ions. The results indicate that b ions produced from acidic peptides can also undergo head-to-tail cyclization, which is the reason for the formation of the non-direct sequence b ions. The b ion spectra derived from the peptides became more complex as the number of acidic residues in the peptides increased. Side chains of glutamic and aspartic acid did not inhibit the cyclization of the b ions. Substantial water elimination was observed in all CID spectra of b 7 and b 8 ions. Finally, the preferential cleavage of glutamic or aspartic acid residues from macrocyclic structures of b ions was also investigated under various collision energy conditions.

a b s t r a c tA study was carried out to examine if the amine ( NH 2 ) group located on the side chains of lysine (K), glutamine (Q), or asparagine (N) residue has any effect on the macrocyclization of b ions even though the N-terminals of the peptides were acetylated. The work utilized the model peptides Ac-KYAGFLVG, Ac-QYAGFLV-NH 2 , and Ac-NYAGFLV-NH 2 . The CID mass spectra of b 7 ions originated from these three peptides exhibited that the macrocyclization still occurred for the lysine containing peptide in spite of the N-terminal of the peptide was acetylated, but was failed to be observed for glutamine and asparagine containing peptides. These current results reveal that the lysine side chain -amine group has been involved in the macrocyclization of the peptide b ions for the N-terminal acetylated peptides and consequently, non-direct sequence b ions were observed in the CID mass spectra. However, due to the amide group on the side chains of the glutamine and asparagine residues, the nucleophilicity of their groups greatly reduced; therefore the scrambling b ions were not detected in their b 7 ion CID mass spectra. In addition, the effect of the lysine position was also studied for series of six isomeric octapeptides such as, Ac-KYAGFLVG, Ac-YKAGFLVG, Ac-YAKGFLVG, Ac-YAGKFLVG, Ac-YAGFKLVG and Ac-YAGFLKVG in order to examine the relationship between the intensities of non-direct sequence b ions and the lysine position in the octapeptide series. The results clearly demonstrated that the most abundant non-direct sequence b ions were observed for the first position of lysine residue in the N-terminal acetylated octapeptide, however, when the lysine residue gets closer to the C-terminal position the relative intensities of the scrambled b ions were greatly decreased.

a b s t r a c tIn this study, we investigated the effect of the second amino acid identity of hexapeptides on gas-phase structures and the proton affinities of N-terminal proline containing b 2 + ions produced from the fragmentation of b 6 + ions under low-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). It should be noted that, among all other fragments, the b 2 + and nominallywere mainly considered in this study. This is a unique example of consecutive cleavage of b 6 + ions which fragments to b 2 + and nominal b 4 + ions. All structural and proton affinity calculations for b 2 + ions were carried out with the B3LYP/6-31+G(d,p) level of theory. The study utilized C-terminal amidated model peptides consisting of PAAAAA-NH 2 and PXAAAA-NH 2 where X is phenylalanine (F), glutamic acid (E), tryptophan (W), and histidine (H) residue. Two main structural isomers of b 2 + ions, namely oxazolone and diketopiperazine, have been considered for the computations. The results demonstrated that the proton affinities of oxazolone isomers of PX are greater than its diketopiperazine isomers. Higher correlation coefficient is calculated if the structure of PX is considered as oxazolone rather than diketopiperazine isomer. Additionally, a linear fit is observed between intensity ratio (PX/AAAA) and calculated proton affinities of PX ions. Additionally, MS/MS results revealed that the relative intensities of b 2 + -PA, PF, and PE-ions are lower compared to the relative intensity of AAAA fragment ion. In contrast, b 2 + -PW and PH-ions have higher relative intensities compared to the AAAA ion. This behavior is explained by the proton affinities of fragment ions computationally.

O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dHO) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC-MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions.

Characterization of ε-N-acetylated lysine containing peptides, one of the most prominent post-translational modifications of proteins, is an important goal for tandem mass spectrometry experiments. A systematic study for the fragmentation reactions of b ions derived from ε-N-acetyllysine containing model octapeptides (K Ac YAGFLVG and YAK Ac GFLVG) has been examined in detail. Collision-induced dissociation (CID) mass spectra of b n (n = 4-7) fragments of ε-N-acetylated lysine containing peptides are compared with those of N-terminal acetylated and doubly acetylated (both ε-N and N-terminal) peptides, as well as acetyl-free peptides. Both direct and nondirect fragments are observed for acetyl-free and singly acetylated (ε-N or N-terminal) peptides. In the case of ε-N-acetylated lysine containing peptides, however, specific fragment ions (m/z 309, 456, 569 and 668) are observed in CID mass spectra of b n (n = 4-7) ions. The CID mass spectra of these four ions are shown to be identical to those of selected protonated C-terminal amidated peptides. On this basis, a new type of rearrangement chemistry is proposed to account for the formation of these fragment ions, which are specific for ε-N-acetylated lysine containing peptides. Consistent with the observation of nondirect fragments, it is proposed that the b ions undergo head-to-tail macrocyclization followed by ring opening. The proposed reaction pathway assumes that b n (n = 4-7) of ε-N-acetylated lysine containing peptides has a tendency to place the K Ac residue at the C-terminal position after macrocyclization/reopening mechanism. Then, following the loss of CO, it is proposed that the marker ions are the result of the loss of an acetyllysine imine as a neutral fragment.

Abstract. In this study, C-terminal protonated dipeptide eliminations were reported for both b 5 and b 4 ions of side chain hydroxyl group (-OH)

No abstract

The gas‐phase fragmentation reactions of the a7 ions derived from glutamine (Q) containing model heptapeptides have been studied in detail with low‐energy collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Specifically, the positional effect of the Q residue has been investigated on the fragmentation reactions of a7 ions. The study involves two sets of permuted isomers of the Q containing model heptapeptides. The first set contains the QAAAAAA sequence, and the second set involves of QYAGFLV sequence, where the position of the Q residue is changed from N‐ to C‐terminal gradually for both peptide series. An intense loss of ammonia from the a7 ions followed by internal amino acid eliminations strongly supports forming the imine‐amides structure via cyclization/rearrangement reaction for all studied a7 ions. This is in agreement with the pioneering study reported by Bythell et al. (2010, 10.1021/ja101556g). A novel rearrangement reaction is detected upon fragmentation of imine‐amide structure, which yields a protonated C‐terminal amidated hexapeptide excluding the Q residue. A possible fragmentation mechanism was proposed to form the protonated C‐terminal amidated hexapeptide, assisted via nucleophilic attack of the side chain amide nitrogen of the Q residue on its N‐protonated imine carbon atom of the rearranged imine‐amide structure. Highlights The gas‐phase fragmentation reactions of a7 ions obtained from protonated model peptides containing glutamine residue were studied by ESI‐MS/MS. A rearranged imine‐amide structure is the predominant even for a7 ions. Novel rearrangement reaction is observed which forms a protonated C‐terminal amidated hexapeptide excluding Q residue upon fragmentation of the imine‐amide structure.