Although N-terminus arginylation was originally thought to be the only site for targeting by ATE1 enzymes, it has recently been discovered that arginylation may also occur in the middle of the peptide chain of a protein. The first discovery of this unprecedented modification occurred when neurotensin, a biological peptide found in the central nervous system, was isolated from cells and it was discovered that arginine was attached to a mid-chain glutamine residue. This discovery changed the view of how arginylation occurs, as this meant that there may be ways to modify and arginylate intact proteins. 
In an effort to determine the prevalence of mid-chain arginylation, a mass spectrometry screen of various peptides was performed. The results from this experiment revealed a plethora of various proteins which contained modified asparagine and glutamine residues present in the middle of their peptide chain, and further studies determined that ATE1 could also be mediating this reaction. Indeed, this discovery changed the biological scope of arginylation by suggesting that arginylation can also occur on fully intact proteins, not just on the N-terminus of protein fragments or pre-processed proteins.