Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein L-isoaspartate O-methyltransferase-deficient mice

Abstract

The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

Figure 1

Enzymatic detection of isoAsp in mouse urinary species via PIMT-catalyzed methylation followed by fluorography of ³H-methyl ester after SDS-PAGE. Isoaspartyl groups in unfractionated (whole) urine were compared to fractionated urine with species less than 10 kDa (<10; peptide fraction) and greater than 10 kDa (=10; protein fraction). The samples were labeled with ³H-methyl groups from ³H-AdoMet catalyzed by rhPIMT and separated by SDS-PAGE. The dried and EN³HANCEd Coomassie-stained gel is shown on the left, and the fluorograph on the right (82 day exposure at -80 °C using Kodak BioMax XAR film). The asterisk (*) denotes the position of auto-methylated rhPIMT.

Figure 2

Extracted ion chromatograms and ETD spectra of MUP1 peptide (50-73), IE⁵²D⁵³NG⁵⁵NFRLFLEQIHVLE⁶⁸NSLVLK. (a) Extracted ion chromatograms of MUP1 peptide and its isomers from wild type and PIMT-deficient mice; (b) ETD spectrum of native MUP1 peptide (50-73); (c,d) The reporter ions of isoAsp (c₃+57, z₆-57) in ETD MS/MS spectra from the selected precursors of above peptide and its isoaspartyl isoforms.

Scheme 1

Formation of isoAsp via deamidation of asparagine or isomerization of aspartic acid and the repair of isoAsp by PIMT. The peptide backbone is highlighted in bold.

Scheme 2

Integrated proteomic workflow of major isoAsp proteins in mouse urine.