Angiotensin I, a model decapeptide, was glycosylated and partially hydrolyzed with HCl (6 N, 80 $^{\circ}C$, 4 h), aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in the partial acid hydrolysate of angiotensin and its glycosylation produc...
Angiotensin I, a model decapeptide, was glycosylated and partially hydrolyzed with HCl (6 N, 80 $^{\circ}C$, 4 h), aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in the partial acid hydrolysate of angiotensin and its glycosylation product mixture by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry enabled sequencing of angiotensin by a combinatorial procedure. MALDI-TOF and electrospray ionization (ESI) tandem mass spectrometric results indicate that both the N-terminal amino group of aspartic acid and the guanidinium group of the second residue arginine are glycosylated.
Angiotensin I, a model decapeptide, was glycosylated and partially hydrolyzed with HCl (6 N, 80 $^{\circ}C$, 4 h), aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in the partial acid hydrolysate of angiotensin and its glycosylation product mixture by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry enabled sequencing of angiotensin by a combinatorial procedure. MALDI-TOF and electrospray ionization (ESI) tandem mass spectrometric results indicate that both the N-terminal amino group of aspartic acid and the guanidinium group of the second residue arginine are glycosylated.
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가설 설정
10. At each step, the molecular weight of the amino acids in the tentative sequence was added and the resulting molecular weight minus the appropriate number of water molecules was compared with the molecular weight of the parent peptide. This match was obtained with the sequence DRVY( )LHPFHL.
5. Truncation of D from (1046.54) left the E/F question unresolved. However, observation of F truncation clearly showed that phenylalanine is the third amino acid from the C-terminus, because F cannot be the N-terminal amino acid.
제안 방법
And the partial sequence informations could be used to elongate the tentative sequence and eventually to construct the full sequence. In this paper, we demonstrate how this combinatorial approach could be used to sequence angiotensin I, a decapeptide, and to investigate the glycosylation site. ESI tandem mass spectrometric investigation of the glycosylation site is also discussed.
Using a small model peptide, angiotensin I, we tested the feasibility ofusing the combinatorial sequencing method for identifying the glycosylation site of a peptide. If partial hydrolysis produced a peptide and its truncation product, the mass difference could be used to obtain a unique combination or a small number of combinations of amino acids.
We note that the analysis of the tandem mass spectra would have been difficult if the sequence information obtained from the MALDI experiments was not available. With the sequence information the combined MALDI-TOF and ESI tandem mass spectrometric data could be used to confirm the involvement of N-terminal D and R in the glycosylation reaction. It would be ofinterest to compare the reactivity of the N-terminal amino group and the guanidinium group using a peptide where R is toward the C-terminus.
대상 데이터
Voyager-DE STR Biospectrometry workstation with delayed extraction and reflectron capability (PE Biosystems, Framingham, MA, USA) was used. It was equipped with a 337 nm nitrogen laser and a 2 m flight tube. Mass spectra were obtained in the positive ion mode.
이론/모형
ESI tandem mass spectrometry was performed using a hybrid quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Q-TOF Ultima Global, Micromass, Manchester, UK). The instrument was operated in the positive ion nanoelectrospray mode; the capillary and cone voltages were +3.
성능/효과
2. Subtraction ofthe masses of 19 x 19 combinations of 2 amino acids from 1296.68 resulted in 6 matches with the observed mass. Subtraction of DL matched 1068.
3. The procedure was repeated starting from 1181.66, second highest in mass among peptides without glucose. Designation of the parent peptide as D(1181.
6. Repeating the procedure starting from 1025.58, tyrosine (Y) was identified, based on the observation of the 763.53 peak, as the fourth amino acid from the N-terminus. The tentative sequence is DRVY( )PFHL.
7. Repeating the procedure starting from 931.52, 926.50, and 912.52 only confirmed the sequence DRVY( )PFHL.
참고문헌 (17)
Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.;Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Comm. MassSpectrom. 1996, 10, 889-896.
Park, D.-H. M.S. Thesis, Seoul National University: Feb 2003.
Fukamizo, T.; Juffer, A. H.; Vogel, H. J.; Honda, Y.; Tremblay, H.;Boucher, I.; Neugebauer, W. A.; Brzezinski, R. J. Biol. Chem.2000, 275, 25633-25640.
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