N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation

Abstract

De novo sequencing is still essential in the identi cation of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. e discrimination is possible based on di erences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N-or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is o en hampered in those derivatives with a xed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton a nity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also e ective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most e ectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. is derivatization approach, which is compatible with both HE-and LE-CID analysis, o ers a straightforward and unambiguous de novo peptide sequencing method.

2,5-Dihydroxyterephthalic Acid: A Matrix for Improved Detection and Imaging of Amino Acids

“…Furthermore, Ile has a methyl group at the β-carbon in its side chain, while Leu has only one hydrogen atom at the β-carbon. The structural difference also makes Ile’s side chain more prone to breaking than Leu’s . Subsequently, the detection performance of DHT, DHB, and CHCA was evaluated using MALDI-MS in positive-ion mode for a mixture containing 20 protein AAs with DHT, DHB, and CHCA as the MALDI matrices, respectively (Figure ).…”

“…The structural difference also makes Ile's side chain more prone to breaking than Leu's. 50 Subsequently, the detection performance of DHT, DHB, and CHCA was evaluated using MALDI-MS in positive-ion mode for a mixture containing 20 protein AAs with DHT, DHB, and CHCA as the MALDI matrices, respectively (Figure 2). The results showed that the DHT matrix detected all individual AAs from the mixed solution, whereas DHB and CHCA only detected 8 and 11 AAs, respectively (Figure 2D).…”

A Facile Method for Preferential Modification of the N-Terminal Amino Group of Peptides Using Triazine-Based Coupling Reagents

“…10) Since Gmb contains a carboxyl group, its introduction can be achieved by formation of an amide bond with the amino group of peptides using condensation reagents. DMT-MM has been widely used for amide bond formation between various compounds, due to its high reactivity superior to other reagents such as carbodiimide or NHS.…”

New Derivatization Strategies for Installation of Fixed, Permanent Charges onto Analytes of Biological and Environmental Concern

“…Grignard synthetic method reaction attempts with reaction conditions and modifications to produce 2,2-dimethyl-5-(5-methylhex-1-yn-3-yl)-1,3-dioxane-4,6-dione (12) following the procedure outlined by Krabbe. 71 (16). Conditions and procedures are modifications of procedures outlined by Bigi, Dumas and Sobrinho.…”

“…15 Another CD reagent that contains a highly ionizable group is 4-amidinobenzoic acid (Aba) as seen in Figure 1.3. 16 This reagent was highly successful at increasing sensitivity and generating d-ions of the peptide to allow differentiation of leucine and isoleucine residues. 16 However, derivatization reactions with peptides occurred overnight at room temperature thus quick analysis of analytes was limited.…”