An electrospray‐ionization—flow‐tube study of H/D exchange in protonated leucine‐enkephalin

Abstract

An electrospray ionization fast flow technique has been employed to study reactions with CH 3 OD and ND 3 , respectively, of the protonated pentapeptide leucine enkephalin, and of protonated tri-and tetrapeptides that make up its backbone structure. Deconvolution of the experimental mass spectral data followed by simulation of the kinetic data by solution of differential equations leads to sets of apparent and site-specific rate constants. On a time scale of several milliseconds, leucine enkephalin undergoes with ND 3 four fast H/D exchanges and one slow exchange. Three equivalent exchanges are observed that are ascribed to the protonated amine group at the N-terminus. Contrary to leucine enkephalin, some of the smaller peptides that do not have tyrosine at the N-terminus are reactive with CH 3 OD, but the reactions are more than two orders of magnitude slower than the ND 3 reactions. These peptides do not sequester the ionizing proton at the N-terminus. The present study demonstrates the capabilities of a fast flow technique in studying gas phase reactions of small biomolecules. It forms part of an effort to understand gas phase conformational changes in proteins through hydrogen-exchange electrospray ionization mass spectrometry.

Energetics and Dynamics of Fragmentation of Protonated Leucine Enkephalin from Time- and Energy-Resolved Surface-Induced Dissociation Studies

“…Analysis of site-specific H/D exchange rates performed by Lifshitz and co-workers suggested that YGGFL is protonated at the N-terminal amino group. 52 Preferential cleavage of the fourth peptide bond requires a transfer of the ionizing proton from the N-terminus to another side of the peptide. This can be explained only based on the secondary structure of the peptide.…”

“…Cumulatively, cleavage of this bond accounts for more than 60% fragmentation of YGGFL. Analysis of site-specific H/D exchange rates performed by Lifshitz and co-workers suggested that YGGFL is protonated at the N-terminal amino group . Preferential cleavage of the fourth peptide bond requires a transfer of the ionizing proton from the N-terminus to another side of the peptide.…”

Candida Cholecystitis in a Diabetic Patient on Chronic Peritoneal Dialysis

“…Although the mass spectrum of protonated YGGFL is relatively simple, much effort has been invested to understand fragmentation processes in detail (Barber et al, 1981; Katakuse & Desiderio, 1983; Gaskell, Reilly, & Porter, 1988; Alexander & Boyd, 1989; Baeten et al, 1989; Meot‐Ner (Mautner) et al, 1995; Vekey, 1996; Schnier et al, 1997; Ustyuzhanin et al, 2001; Rakov, Borisov, & Whitehouse, 2004; Laskin, 2006a; Campbell et al, 2007; Allen et al, 2008). YGGFL has various protonation sites, the N ‐terminal amino group, the C‐terminal carboxylic acid, the amide nitrogen and oxygen atoms.…”

“…YGGFL has various protonation sites, the N ‐terminal amino group, the C‐terminal carboxylic acid, the amide nitrogen and oxygen atoms. Based on H/D exchange experiments, it has been established that the most favorable site is the N ‐terminal amino group (Ustyuzhanin et al, 2001). This finding was confirmed with experiments and with high‐quality calculations (Polfer et al, 2007), which showed that protonation at the amide oxygen is approximately 33.5 kJ/mol less favored.…”

9 Organic gas phase ion chemistry

“…H/D exchange reactions of protonated peptides with reagents such as CH 3 OD and ND 3 have also been studied. 353,354 On the short timescale of these experiments the following observations were made: (i) reaction of doubly protonated bradykinin with ND 3 results in three fast H/D exchanges and one slow exchange, while exchanges with CH 3 OD are nearly 2 orders of magnitude slower; (ii) the more efficient exchange of doubly protonated des-Arg 9 -bradykinin with ND 3 is accompanied by formation of collisionally stabilized [M ϩ 2H ϩ ND 3 ] 2ϩ complexes; and (iii) protonated leucine enkephalin undergoes four fast H/D exchanges and one slow exchange with ND 3 but is unreactive towards CH 3 OD. 355 Using CID to produce b and y sequence ions provides information about the sites of H/D exchange.…”