Method of Isotope labeling |
genetic engeneering-based in vitro labeling |
cell culture-based in vivo (metabolic) labeling |
|
PTM in a reference RNA |
no |
yes |
Metabolically labeled RNA undergoes PTMs in the cell |
Detection of PTM |
data dependent |
prediction of MS shift reqired |
|
Detection of pseudouridine |
shift of retention time in LC/chemical modification, e.g. cyanoethylation |
metabolic labeling in the presence of 5,6-D2 uridine |
|
Quantitation of PTM |
absolute quantitation (to estimate the absolute stoichimetry of PTM ) |
relative quantitaion (to estimate the relative changes in PTM) |
|
Comprehensive PTM analysis |
possible |
difficult |
In vivo labeling method cannot detect "unknown" PTMs with unpredictable MS shifts |