AACT for a 72-hour (nontreated versus ATRA-treated) sample accounting for the quantification and identification of eEF1A-1. (A) The averaged time-of-flight (TOF) MS from C18 high-performance liquid chromatography (HPLC) retention times of 33.5 to 34.2 minutes accounting for tryptic peptide EHALLAYTLGVK. The monoisotopic m/z value of 657.85 represents the +2 charged EHALLAYTLGVK peptide produced in nontreated NB4, and 662.38 represents the EHALLAYTLGVK peptide from ATRA-treated NB4 grown in 99% Leu-d3 (116.1 Da) media. Comparison of the relative intensities of the 2 monoisotopic peaks (657.85 versus 662.38) yields a ratio (ATRA treated to nontreated) of 0.32, implying down-regulation of 3.1-fold via ATRA treatment. (B) The automated MS/MS fragmentation pattern of 657.85. The peptide sequence produced from MS/MS fragmentation yielded the identification of eEF1A-1. A few fragmentation b- and y-ions are labeled for comparison with MS/MS data from 662.38. (C) The automated MS/MS fragmentation pattern of 662.38. A 3.02 Da difference exists for each fragmented peptide containing leucine as illustrated by y7, y8, and y9, which yield m/z values of 754.4, 870.5, and 986.6, respectively. Fragments that do not contain leucine are identical to the MS/MS data for 657.85, as demonstrated by b2 and b3.
