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.

The collision energy or collision voltage necessary to obtain 50% fragmentation (characteristic collision energy/voltage, CCE or CCV) has been systematically determined for different types of molecules [poly(ethylene glycols) (PEG), poly(tetrahydrofuran) (PTHF), and peptides] over a wide mass (degrees of freedom) range. In the case of lithium-cationized PEGs a clear linear correlation (R(2) > 0.996) has been found between CCE and precursor ion mass on various instrument types up to 4.5 kDa. A similar linear correlation was observed between CCV and the mass-to-charge ratio. For singly and multiply charged polymers studied under a variety of experimental conditions and on several instruments, all data were plotted together and showed correlation coefficient R(2) = 0.991. A prerequisite to observe such a good linear correlation is that the energy and entropy of activation in a class of polymers is likely to remain constant. When compounds of different structure are compared, the CCV will depend not only on the molecular mass but the activation energy and entropy as well. This finding has both theoretical and practical importance. From a theoretical point of view it suggests fast energy randomization up to at least 4.5 kDa so that statistical rate theories are applicable in this range. These results also suggest an easy method for instrument tuning for high-throughput structural characterization through tandem MS: after a standard compound is measured, the optimum excitation voltage is in a simple proportion with the mass of any structurally similar analyte at constant experimental conditions.