• Mass Spectrometry Letters
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• 제12권2호
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• pp.41-46
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• 2021

Collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) are the widely used fragmentation technique in mass spectrometry-based proteomics studies. Understanding the fragmentation pattern from the tandem mass spectra using statistical methods helps to implement efficient spectrum analysis algorithms. The study characterizes the frequency of occurrence of multi-charged fragment ions and their neutral loss events of doubly and triply charged peptides in the CID and HCD spectrum. The dependency of the length of the fragment ion on the occurrence of multi-charged fragment ion is characterized here. Study shows that the singly charged fragment ions are generally dominated in the doubly charged peptide spectrum. However, as the length of the product ion increases, the frequency of occurrence of charge 2 fragment ions increases. The y- ions have more tendencies to generate charge 2 fragment ions than b- ions, both in CID and HCD spectrum. The frequency of occurrence of charge 2 fragment ion peaks is prominent upon the dissociation of the triply charged peptides. For triply charged peptides, product ion of higher length occurred in multiple charge states in CID spectrum. The neutral loss peaks mostly exist in charge 2 states in the triply charged peptide spectrum. The b-ions peaks are observed in much less frequency than y-ions in HCD spectrum as the length of the fragment increases. Isotopic peaks are occurred in charge 2 state both in doubly and triply charged peptide's HCD spectrum.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.399-405
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• 1998

From the sequence alignment of various non-ribosomal peptide synthetases, several motifs of highly conserved sequences have been identified within each domain of peptide synthetases. We designed PCR primers based on the highly conserved nucleotide sequences to amplify and isolate a ∼7.2-kb DNA fragment of the Bacillus subtilis 713 which was isolated and reported to produce an antifungal peptide compound. Nucleotide sequence analysis of 4.8 kb of the predicted amino acids revealed significant homology to various peptide synthetases over the whole sequence and also revealed two amino acid-activating domains with highly conserved Core 1 to Core 6 and spacer motif. This suggests that the isolated DNA fragment is part of a peptide synthetase gene for antifungal peptide.

• Parasites, Hosts and Diseases
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• 제47권1호
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• pp.31-36
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• 2009

Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.

• Archives of Pharmacal Research
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• 제24권5호
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• pp.446-454
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• 2001

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.276-281
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• 1999

In order to determine the functional region of the antifungal protein (AFP) isolated from Aspergillus giganteus responsible for growth inhibitory activity and the promotion of phospholipid vesicle aggregation, overlapping peptides covering the complete sequence of AFP were synthesized. The antibiotic activity against bacterial, fungal, and tumor cells, and the vesicle-aggregation activity of the synthetic peptides were investigated. The AFP functional sequence responsible for antibiotic and vesicle-aggregation activity was determined to be located within the region between AFP residues 19 to 32. AFP (19-32) exhibited an a-helical conformation in a cell membrane-like environment. AFP (19-32) displayed potent antibiotic activity against bacterial, fungal, and tumor cells without peptide toxicity as indicated by hemolysis. Accordingly, AFP (19-32) could be used as a good model for the design of effective antibiotic agents with powerful antibiotic activity yet without any cytotoxic effects against the host organism.

• 생명과학회지
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• 제31권1호
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• pp.83-89
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• 2021

개불 라이소자임(Uu-ilys)은 개불(Urechis unicinctus)로부터 정제된 무척추형 라이소자임으로 병원균에 대한 방어에 주요하게 작용하는 선천성 면역 물질이며, 비효소적 항균 활성을 가지고 있어 항균 활성을 지닌 유도체의 개발 가능성을 가지고 있다. 본 논문은 개불 라이소자임에서 유래된 항균 활성을 가지는 유도체의 디자인과 생산을 기술하고 있다. 여러 항균성 펩타이드(antimicrobial peptide, AMP) 데이터베이스에서 제공하는 항균성 펩타이드 예측 도구를 사용하여 개불 라이소자임에서 항균 활성을 가지는 부위를 예측하였다. 개불 라이소자임 C-말단의 절편이 항균 활성을 나타낼 것으로 예측되었으며, 이 펩타이드는 C-말단 절편, Uu-ilys-CF라 명명하였다. Uu-ilys-CF은 이형 발현 시스템인 TrxA-Uu-ilys-CF 퓨전 단백질을 사용하여 생산하였다. 만들어진 퓨전 단백질은 브롬화시안을 사용하여 메티오닌 잔기를 절단하였으며, 절단된 Uu-ilys-CF은 고성능액체크로마토그래피와 역상 컬럼인 CapCell-Pak C18을 사용하여 분리되었다. Uu-ilys-CF의 항균 활성을 조사하기 위해서 여러 균주(그람양성균 4개, 그람음성균7개, 진균 1개)를 사용하였다. Uu-ilys-CF의 항균 활성은 살모넬라에서 가장 높은 반응을 보였다. 비록 Uu-ilys-CF는 진균에 활성을 나타내지 않았지만, 사용한 균주들에서 넓은 범위의 항균 활성을 나타내었다.

• Mass Spectrometry Letters
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• 제10권2호
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• pp.50-55
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• 2019

The fragmentation statistics of ion trap CID (Collision-Induced Dissociation) spectra using 87,661 tandem mass spectra of doubly charged tryptic peptides are analyzed here. In contrast to the usual method of using intensity information, the frequency of occurrence of fragment ions, with respect to the position of the cleavage site and the residues at these sites is studied in this paper. The analysis shows that the frequency of occurrence of fragment ion peaks is more towards the middle of the peptide than its ends. It was noted that amino acid with an aromatic and basic side chain at N- & C- terminal end of the peptide stimulates more peaks at the lower end of the spectrum. The residue pair effect was shown when the amide bond occurs between acidic and basic residues. The fragmentation at these sites (D/E-H/R/K) stimulates the generation of the y-ion peak. Also, the cleavage site H-H/R/K stimulates the generation of b-ions. K-P environment in the peptide sequence has more tendency to generate y-ions than b-ions. Statistical analysis helps in the visualization of the CID fragmentation pattern. Cleavage pattern along the length of the peptide and the residue pair effects, enhance the knowledge of fragmentation behavior, which is useful for the better interpretation of tandem mass spectra.

• Genomics & Informatics
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• 제4권2호
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• pp.65-70
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• 2006

Peptide mass mapping is the matching of experimentally generated peptides masses with the predicted masses of digested proteins contained in a database. To identify proteins by matching their constituent fragment masses to the theoretical peptide masses generated from a protein database, the peptide mass fingerprinting technique is used for the protein identification. Thus, it is important to know the theoretical mass distribution of the database. However, few researches have reported the peptide mass distribution of a database. We analyzed the peptide mass distribution of non-redundant protein sequence database in the NCBI after digestion with 15 different types of enzymes. In order to characterize the peptide mass distribution with different digestion enzymes, a power law distribution (Zipfs law) was applied to the distribution. After constructing simulated digestion of a protein database, rank-frequency plot of peptide fragments was applied to generalize a Zipfs law curve for all enzymes. As a result, our data appear to fit Zipfs law with statistically significant parameter values.

• Bulletin of the Korean Chemical Society
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• 제32권12호
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• pp.4337-4340
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• 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

• 대한수의학회지
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• 제48권3호
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• pp.251-258
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• 2008

The neurotoxicity of C-terminal fragments of amyloid precusor protein (CT) is known to play some roles in Alzheimer's disease progression. In this study, we investigated the effects of the recombinant C-terminal 105 amino acid fragment of amyloid precusor protein (CT105) on cholinergic function using CT105-injected rat. To study the effects of CT105 on septohippocampal pathway, choline acetyltransferase (ChAT) positive neurons were examined in the medial septum and in the diagonal band after an injection of CT105 peptide into the lateral ventricle. Immunohistological analysis revealed that the number of ChAT-immunopositive cells decreased significantly in both medial septum and diagonal band. In addition, CT105 decreased ChAT-immunopositive cells in the hippocampal area, particulary in the dentate gyros. To study the effect of amyloid beta peptide ($A{\beta}$) and CT105 on the cholinergic system, each peptide was injected into the left lateral ventricle, and acetylcholine (ACh) levels were monitored in hippocampus. ACh level in the hippocampal area was reduced to 60% of control level in $A{\beta}$-treated group, and the level was reduced to 15% of control level in CT105-treated group, at one week after the injection. ACh level was further reduced to 35% of control in $A{\beta}$-treated group, whereas the level was slightly increased to 30% of control in CT105-treated group at 4 weeks after the injection. Taken together, the results in the present study suggest that CT105 impairs the septohippocampal pathway by reducing acetylcholine synthesis and release, which results in damage of learning and memory.