Characteristic ions of the tryptic fragments of eEF1A proteins detected by mass spectrometry.

HA-eEF1A proteins expressed in T. brucei were purified, digested with trypsin and subjected to nano-LC-MS/MS as described in Materials and Methods. Purified carboxy-terminally His6x-tagged S. cerevisiae eEF1A was treated prior to nano-LC-MS/MS the same way as for HA-tagged eEF1A proteins. Tryptic fragments containing the site of potential EPG attachment E362, E298/E372, E301/E374 of domainII/domain III from T. brucei, S. cerevisiae and H. sapiens eEF1A, respectively (all marked with an asterisk) are shown with their corresponding [M+H]+, [M+H]2+ and [M+H]3+ ions. The last column indicates the presence (+) or absence (−) of EPG modifications based on ion data.n.d., not detected.−,not present.Ox, oxidation.a,described in [15].b,expressed as HA-tagged protein in T. brucei.c,expressed as His6x-tagged protein in S. cerevisiae.d,the relative intensities of the [M+H]+ ions of the EPG-modified (m/z 1020.465) and unmodified (m/z 823.420) tryptic peptides suggest that >95% of T. brucei eEF1A is modified with EPG (see [15]).