서강대학교

초록/요약

Part Ⅰ. One-Step Free Radical Initiated Backbone Dissociation in Positive-Ion CID of peptides-conjugated with p-TEMPO-Benzyl Succinic Acid Radical Initiator. 4-(2,2,6,6,-tetramethylpiperidin-1-oxyl) methyl benzyl succinic acid N-hydroxysuccinimide, so-called p-TEMPO-Bz-Sc-NHS) was designed and synthesized to achieve free radical initiated peptide sequencing (FRIPS) in one-step MS/MS of low energy positive CID. The p-TEMPO-Bz-Sc- radical initiator was conjugated with the peptides, including Angiotensin Ⅱ (DRVYIHPF), kinetensin (IARRHPYFL), glycoprotein Ⅱb fragment (296-306) (TDVNGDGRHDL), and des-Pro2-bradykinin (RPPGFSPFR). The p-TEMPO-Bz-Sc-C(O)-peptides were analyzed at MS/MS and MS3 for FRIPS on positive-ion mode. The C-O cleavage of p-TEMPO-Bz-Sc-C(O)-peptides leads to dissociation to p-TEMPO radical and •Bz-Sc-C(O)-peptides radical. And then a beta hydrogen (Hβ) abstraction of peptide backbone and radical migration results in the dissociation of peptide backbone which is consistent with the previously studied the dissociation mechanism of o-TEMPO-FRIPS. The fragment ions that are observed in 1-step collisional activation of p-TEMPO-Bz-Sc-C(O)-peptides were mainly a-, c-, x-, and z-type ions and small mass losses. It was confirmed that this procedure also follows the radical driven peptide backbone dissociation. The p-TEMPO-Bz-Sc- radical initiator inserted succinic acid (Sc) between TEMPO and peptide was implemented to the structural flexibility and improved access to beta hydrogen of peptide backbone by the cyclization of the benzyl radical. The energetic interpretation using survival fraction depending on the applied NCE ascertained the difference of FRIPS between p-TEMPO-Bz-Sc- and o-TEMPO-Bz- radical initiator. As the results, the energetic effect of thermodynamic entropy by loose transition state affects the dissociation of p-TEMPO-Bz-Sc-C(O)-peptides in FRIPS mechanism. The application of p-TEMPO-Bz-Sc- radical initiator can improve duty-cycle and the one-step collisional activation of protonated peptide precursor ion could be implemented in proteomics through FRIPS. Part Ⅱ. Side chain cleavage in TEMPO-assisted free radical initiated peptide sequencing (FRIPS): Amino acid composition information. In tandem mass spectrometry, side chain losses are known to provide information on the presence of certain amino acid residues, which can improve peptide identification. In the present study, I examined whether or not side chain losses also occur in free radical initiated peptide sequencing mass spectrometry (FRIPS MS). All four of the peptides examined, e.g., ALPMHIR, YRPPGFSPFR, RPPGFSPYR, and YGGFLRRIRPKLK, examined here showed extensive small group losses in the so-called (M•-X) region. Furthermore, small group loss peaks accompanying backbone fragmentations were also extensively observed. The observed small group losses, either radical or neutral side chain losses, confirmed the presence of certain amino acids, even exhibiting signature peaks for isoleucine and leucine. In addition, mechanisms for small group losses in FRIPS MS are proposed.