Leucine enkephalin--a mass spectrometry standard

The present article reviews the mass spectrometric fragmentation processes and fragmentation energetics of leucine enkephalin, a commonly used peptide, which has been studied in detail and has often been used as a standard or reference compound to test novel instrumentation, new methodologies, or to tune instruments. The main purpose of the article is to facilitate its use as a reference material; therefore, all available mass spectrometry-related information on leucine enkephalin has been critically reviewed and summarized. The fragmentation mechanism of leucine enkephalin is typical for a small peptide; but is understood far better than that of most other compounds. Because ion ratios in the MS/MS spectra indicate the degree of excitation, leucine enkephalin is often used as a thermometer molecule in electrospray or matrix-assisted laser desorption ionization (ESI or MALDI). Other parameters described for leucine enkephalin include collisional cross-section and energy transfer; proton affinity and gas-phase basicity; radiative cooling rate; and vibrational frequencies. The lowest-energy fragmentation channel of leucine enkephalin is the MH(+)  → b(4) process. All available data for this process have been re-evaluated. It was found that, although the published E(a) values were significantly different, the corresponding Gibbs free energy change showed good agreement (1.32 ± 0.07 eV) in various studies. Temperature- and energy-dependent rate constants were re-evaluated with an Arrhenius plot. The plot showed good linear correlation among all data (R(2)  = 0.97), spanned over a 9 orders of magnitude range in the rate constants and yielded 1.14 eV activation energy and 10(11.0)  sec(-1) pre-exponential factor. Accuracy (including random and systematic errors, with a 95% confidence interval) is ±0.05 eV and 10(±0.5)  sec(-1), respectively. The activation entropy at 470 K that corresponds to this reaction is -38.1 ± 9.6 J mol(-1)  K(-1). We believe that these re-evaluated values are by far the most accurate activation parameters available at present for a protonated peptide and can be considered as "consensus" values; results on other processes might be compared to this reference value.