Toward proteome-scale identification and quantification of isoaspartyl residues in biological samples.
Deamidation of asparaginyl and isomerization of aspartyl residues in proteins produce a mixture of aspartyl and isoaspartyl residues, the latter being involved in protein aging and inactivation. Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are known to be able to distinguish the isoaspartyl peptides by unique fragments of cn* + 58.0054 (C2H2O2) and z(l-n)-56.9976 (C2HO2), where n is the position of the aspartyl residue and l is the peptide length. In the present study, we tested the specificity of isoAsp detection using the accurate masses of the specific fragments. For this purpose, we analyzed 32 whole and partial proteomes obtained from human cells as well as tissue samples and identified by ECD 466 isoaspartyl peptide candidates. Detailed inspection revealed that many of these candidates were unreliable. To increase the isoAsp detection specificity, additional criteria had to be used, for example, adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak. Most stringent filtering of candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates for l-isoaspartyl methyltransferase, an important repair enzyme converting isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.
Yang
Hongqian
H
Division of Molecular Biometry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden.
Fung
Eva Y M
EY
Zubarev
Alexander R
AR
Zubarev
Roman A
RA
D000544:Alzheimer Disease; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000032:analysis; D002460:Cell Line; D005583:Fourier Analysis; D006801:Humans; D026581:Isoaspartic Acid / Q000031:analogs & derivatives / Q000032:analysis; D007536:Isomerism; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D026581:Isoaspartic Acid; D010455:Peptides; D011506:Proteins; D001224:Aspartic Acid