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.

Antimicrobial peptides (AMPs) are a diverse group of small, naturally occurring molecules that orchestrate the innate immune response of various organisms, from microorganisms to humans. Characterized by their broad-spectrum activity against bacteria, fungi and viruses, AMPs are increasingly recognized for their potential as novel therapeutic agents in the face of rising antibiotic resistance. Here, we present several newly designed AMPs, one of which, DTN6, exerts significant activity against several organisms with MIC values as low as 0.5 µg/mL. The D-TN6 peptide influences both bacteria and yeasts. Scanning electron microscopy and transmission electron microscopy results showed that the bacterial membrane is affected by D-TN6, which is resistant to proteases and is effective against antibiotic-resistant pathogens with hemolytic activity and low toxicity. The D-TN6 peptide is effective in vivo against standard S. aureus strains in wounds. Thus, D-TN6 is a potent antibiotic candidate with a broad spectrum of activity. Overall, AMPs are a promising tool for the development of next-generation antimicrobial agents that could mitigate global health threats posed by multidrug-resistant pathogens.

A 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 ε-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.TUBITAK (109T430); Mass Spectrometry Facilit

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/dH₂O) 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.

In 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 b2 + ions produced from the fragmentation of b6 + ions under low-energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). It should be noted that, among all other fragments, the b2 + and nominally b4 + (AAAA) ions ([M+H]+ → b6 + → b2 + (PX+) + b4 + (AAAA+) were mainly considered in this study. This is a unique example of consecutive cleavage of b6 + ions which fragments to b2 + and nominal b4 + ions. All structural and proton affinity calculations for b2 + 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-NH2 and PXAAAA-NH2 where X is phenylalanine (F), glutamic acid (E), tryptophan (W), and histidine (H) residue. Two main structural isomers of b2 + 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 b2 +-PA, PF, and PE- ions are lower compared to the relative intensity of AAAA fragment ion. In contrast, b2 +-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.Scientific and Technological Research Council of Turkey (111T935/112T558); State Planning Organization, DP

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine associated with TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), which play important roles in several inflammatory diseases. There is a growing interest in developing alternative molecules that can be used as TNF blockers. In this study, we focused on TNF-α-, TNFR1-, and TNFR2-mimicking peptides to inhibit TNF-α receptor binding in various ways. Six peptides (OB1, OB2, OB5, OB6, OB7, and OB8) were developed to bind TNFR1, TNFR2, and TNF-α. OB1 and OB2 bound to TNF-α with lower K_d values of 300 and 46.7 nM, respectively, compared to previously published sequences. These synthetic peptides directly and indirectly inhibited TNF-α in vitro without cytotoxicity to L929 cells, and OB1 significantly inhibited apoptosis in the presence of hTNF-α. Peptides developed in this study may prove to be useful for therapeutic inhibition of TNF-α.