Chemo-enzymatic detection of protein isoaspartate using protein isoaspartate methyltransferase and hydrazine trapping.
Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.
Alfaro
Joshua F
JF
Department of Chemistry, Washington State University, Pullman, Washington 99164, USA.
Gillies
Laura A
LA
Sun
He G
HG
Dai
Shujia
S
Zang
Tianzhu
T
Klaene
Joshua J
JJ
Kim
Byung Ju
BJ
Lowenson
Jonathan D
JD
Clarke
Steven G
SG
Karger
Barry L
BL
Zhou
Zhaohui Sunny
ZS
D002846:Chromatography, Affinity; D006834:Hydrazines / Q000737:chemistry*; D026581:Isoaspartic Acid / Q000032:analysis*; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D006834:Hydrazines; D026581:Isoaspartic Acid; D011506:Proteins; C029424:hydrazine; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase