Volumetric analysis of unmodified and modified amino acids and its application to the detection of post-translational modifications (PTMs) with a nanopore

Recently a method was proposed for unambiguous identification of an amino acid (AA) in the bulk or at the single molecule (SM) level with a single binary measurement based on the superspecificity property of transfer RNAs (tRNAs) (doi: 10.36227/techrxiv.19318145.v3). The present report looks at detection of post-translational modifications (PTMs) in single AAs after their identification through their cognate tRNAs. The volumes of AAs with PTMs are computed from crystallographic data for all 20 proteinogenic AAs and three types of PTMs (methylation, acetylation, and phosphorylation). The ratio of the spatial volume of a modified AA to that of an unmodified one is then used to assign a PTM to the former. An experimental procedure to obtain these ratios for different PTMs with a nanopore of matching diameter is proposed. AA is freed following its identification via its cognate tRNA as in the procedure mentioned above, separated by a nanofilter, and translocated through a nanopore under electrophoresis. The ratio of the blockade level due to a modified AA to that due to the unmodified AA is compared with the theoretical ratio and a PTM assigned. Because the AA has been unambiguously determined by its cognate tRNA, PTM assignment is horizontal across the set of PTMs, thus the other 19 AA types are not involved in PTM assignment. Computational results are presented for 49 PTMs covering the three types mentioned above. Unlike most methods based on mass spectrometry this is a de novo method that does not require prior knowledge about the parent protein, nor does it use any sequence information from a proteome database.