단백질 정량분석을 위한 지방족 동중체 질량 표지
- 단백질 정량분석을 위한 지방족 동중체 질량 표지
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- Mass-balanced 1H/2H-isotope dipeptide tag (MBIT) is diversified as aliphatic tags for protein quantification. Two kinds of aliphatic MBITs are developed: One is based on the N-acetyl-Xxx-Ala dipeptide, where Xxx represents an artificial amino acid with a linear alkyl side chain from C2H5 to C8H17 (aliphatic C2-C8 tags). The other is based on the N-acyl-Ala-Ala dipeptide, where N-acyl group contains a linear alkyl chain from C2H5 to C8H17 (acyl C2'-C8' tags). Both MBITs provide 2-plex quantitation signals with a 3-Da space. Cn-tags incorporate H/D-isotopes in the methyl groups of N-acetyl and Ala, whereas Cn'-tags encode H/D-isotopes in the two side chains of Ala-Ala. MBIT-linked peptides appear as a single peak in the mass spectra, and yet report quantitation signals separated by 3 Da in the tandem mass spectra. MBITs use N-succinimidyl ester as a linker, which is reactive toward the primary amines of peptides. First, Cn-tags are presented in chapter 2. C2-C5 tags are prepared by solid-phase synthesis, whereas C6-C8 tags are synthesized by olefin metathesis. Relative abundances of quantitation signals from MBIT-linked angiotensin II are characterized using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Elution profiles of MBIT-linked bradykinin on the reverse-phase liquid chromatography (RPLC) are monitored by electrospray ionization (ESI) quadrupole ion trap (Q-trap) MS. MBIT-linked angiotensin II yields 2-plex quantitation signals as well as the sequence ions in similar abundances in the tandem mass spectra. MBIT-linked bradykinin co-migrates in LC. As the length of alkyl side chain increases, C2-C8 tags show a stepwise increase in both the LC retention time and relative abundance of quantitation signals. In addition, the quantitation linearity is good in a 15-250 fmol range of peptides. The multiplexing capability of Cn-tags is demonstrated by applying three different tags (C6-C8 tags) to the quantification of yeast heat shock proteins expressed under four different physiological conditions. Quantification results from nanogram-scale samples using Cn-tags are in excellent agreement with those from microgram-scale samples using gel-based optical imaging. Secondly, Cn'-tags are introduced as alternatives for Cn-tags. C2'- C8' tags are synthesized by solid-phase synthesis. The performances of Cn'-tags are compared with those of Cn-tags using LC-ESI Q-trap and MALDI-TOF/TOF. Effects of alkyl substituents on LC elution profiles and fragmentation patterns of peptides are compared. MBIT-linked bradykinin are separated by nano LC and analyzed by ESI Q-trap in multiple-reaction monitoring (MRM) mode. Cn'-tags also result in the co-elution of tagged peptides. The elution time of MBIT-linked bradykinin is not affected by the location of deuterium, but affected by the length and location of the alkyl group. Four different model peptides (angiotensin II, bradykinin, [Glu1]-fibrinopeptide B, and leucine enkephalin-Arg) are labeled with both Cn'- and Cn-tags, and analyzed with MALDI MS/MS. MALDI results show that relative abundances of quantitation signals (aS and bS) vary with the length and location of the alkyl group. In the cases of Cn'-tags, bS signals are stronger than aS signals, although both aS and bS appear in similar abundances in the cases of Cn-tags. Although Cn'- and Cn-tags are similar in their chemical properties, they result in the different branching ratio of aS/bS. Lastly, the branching ratio of aS/bS is examined by ab initio calculations at the density functional theory (DFT) level. The geometry of the bS ion (protonated oxazolone) is optimized, and those of the aS ion and the transition state are also optimized. All of the bS and aS ions and the transition state are considered to have the same conformation, regardless of the length and location of the alkyl group. From theory, the energy level and activation barrier are calculated. Theory suggests that the alkyl substituents on the N-acyl group stabilize the bS ion but destabilize the transition state, thereby increasing the activation barrier, whereas those on the amino acid side chain stabilize the transition state, thus decreasing the activation barrier.
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