Tubulin is arginylated at E77 on ⍺-tubulin and D74 on β-tubulin in cells. (A) Western blot of microtubule purification steps for mass spectrometry analysis of arginylation. Total protein (top, red); tubulin (bottom, green). (B) Summary of the average ratio of arginylated to total peptide intensity, posterior error probability, and location probability for each arginylation site identified. (C) Sequence alignment of ⍺-tubulin (mouse Tuba1b) and β-tubulin (mouse Tubb5) in the vicinity of the arginylated sites, with the identified peptides underlined and the arginylated residues highlighted in red. (D and E) Structural modeling of the tubulin dimer (PDB ID: 1JFF) showing the location of ⍺E77 (D) and βD74 (E) (highlighted in yellow). Insets show the enlarged regions around the arginylated sites in the microtubule (PDB ID: 3J6F) to illustrate the interactions ⍺E77 and βD74 participate in. Structures are from the RSCB PDB (http://RSCB.org), PDB IDs 1JFF and 3J6F. (F) Extracted ion chromatograms of chemically synthesized non-arginylated and arginylated ⍺-tubulin peptides: CDLEPTVIDEVRTG (top) and CDLEPTVIDE(Arg)VRTG (bottom), showing that the peaks for these two peptides have similar intensities and their abundance can be compared directly in a physiological sample assuming they were not differentially affected by signal suppression from co-eluting peptides. Red boxes indicate the area (AA) and base peak (BP) of the major product. (G) Estimation of the percentage of the peptide AVFVDLEPTVIDEVR that is arginylated at E77 derived from peak intensities in the physiological samples. Source data are available for this figure: SourceData F2.