Extended Data Fig. 9: LC–MS-based parallel reaction monitoring (PRM) analysis of tryptic digest of purified human canonical or non-canonical Fc–APRIL.
From: APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans
a–c, Raw data were analysed using Skyline software and extracted product ion chromatograms (XICs) are shown either in the form of peaks (top) or total sum of integrated product ion areas (bottom) for the three selected peptides EEQYNSTYR (Fc part) (a), LNLSPHGTFLGFVK (tryptic C terminus APRIL) (b) and LNLSPHGTFLGFVKL (miscleaved tryptic C terminus APRIL) (c). MS2 fragment ion spectra for the selected peptide precursor ions are illustrated at bottom right. Although the peptide shown in a is representative for comparable injection amounts of canonical versus non-canonical Fc–APRIL, the C-terminal miscleaved full tryptic peptide shown in c is undetectable in non-canonical APRIL. Relative abundances are given in arbitrary units. Right, FASTA sequence of Fc–APRIL with selected tryptic peptide sequences highlighted in blue or red. Note the different scales in b (109) and c (106). d, Structure of human c-APRIL highlighting the importance of the C terminus for the folding of the different forms (canonical and non-canonical) of APRIL. The representation based on protein data bank accession number 1XU1 highlights the last two C-terminal amino acids (Lys232, Leu233). The N-terminal amino acid of the TNF homology domain (His98) and Asp142 are also shown. All of these residues are conserved in mouse APRIL and human APRIL, although the sequence surrounding Asp142 is different in mouse and human. The C-terminal carboxylic group of Leu233 is very close to His98 of the same monomer (3.6 Å, 4.1 Å and 3.4 Å in the three monomers) and also very close to His98 of the neighbouring monomer (4.3 Å, 3.8 Å and 3.8 Å). Thus, His98 and the carboxylic group or Leu233 seem to form a ring of six salt bridges at the top surface of APRIL. In addition, Lys232 contacts Asp142 (4.3 Å, 4.3 Å and 5.7 Å in the three mouse APRIL monomers), and is only 3.2 Å from Asp142 in human APRIL (PDB accession number 4ZCH).
