Figure 2.

MALDI-TOF Analysis of the AAV5 VP1, VP2, and VP3 Capsid Proteins Stoichiometry

(A) Amino acid sequence of the AAV5 capsid with VP1 unique N termini marked in green, unique VP2 in red, and common VP3 C termini in black. The downward arrows indicate the respective proteins. Tryptic peptides selected for MS analysis are underlined and shown below their respective observed masses. (B) MALDI-TOF-MS spectrum of all tryptic peptides of rAAV5 digested in H₂¹⁸O. The red highlighted peptide is one representative out of three analyzed. (C) Two overlaid MALDI-TOF MS spectra of the same tryptic peptide TWVLPSYNNHQYR originating from the VP1 gel band digested with trypsin prepared in ¹⁶O water (red trace) or from the VP3 gel band digested with trypsin prepared in ¹⁸O water (blue trace). ¹⁸O water incorporates two ¹⁸O atoms on the C terminus of the peptide, thus shifting the mass by 4 atomic mass units (amu). These digestion products were spotted/analyzed separately and the spectra overlaid to show the complete incorporation of two ¹⁸O into the VP3 peptide. (D) Isotopic “fingers” of the same peptide derived from VP1 or VP3 after the digestion products were mixed at 1:1 ratios to calculate the relative content. See also Tables S2 and S3 and Figure S3.