MALDI-MS analysis
MALDI-TOF MS was carried out with a 4800 MALDI-TOF/TOF mass spectrometer from Applied Biosystems (Framingham, MA, USA). All mass spectra were obtained by averaging 2500 laser shots from each sample well in the positive-ion mode. The instrument was controlled using the 4000 series Explorer software (version 3.6; Applied Biosystems). Data were processed by using Data Explorer software (version 4.8; Applied Biosystems). The calibration of the instrument in the MS and MS/MS modes were carried out by using the ions derived from a mixture of several synthetic peptides (Angiotensin I, 1296.685; Dynorphin A, 1603.991; ACTH (1-24), 2932.588; beta-endorphin, 3463.830), and several product ions derived from angiotensin I, respectively. Ordinary MS/MS spectra were obtained with a fully automatic workflow, in which the metastable suppressor and CID gas (air) were both activated.
Peptides dissolved in water/acetonitrile/TFA solutions (0.5 μL) were mixed with the (0.5 μL) matrix solution, (the supernatant of a 50 % acetonitrile solution saturated with α–cyano-4–hydroxycinnamic acid), and then air dried on the flat surface of a stainless steel plate. The ions were generated by irradiating the sample area with the output of an Nd/YAG laser at a wavelength of 355 nm, and the resulting generated ions were accelerated at a 20 kV potential in the ion source.