posted on 2021-07-09, 15:39authored byMiyang Li, Ting-jia Gu, Xiaorong Lin, Lingjun Li
As
one of the most important post-translational modifications,
glycosylation plays a pivotal role in many essential physiological
functions, including cell recognition, signaling, and immune response.
Thus, various qualitative and quantitative analytical strategies for
glycomic profiling have been developed in recent decades. However,
while extensive efforts have been devoted to the analysis of N-glycans, high-throughput quantitative analysis of O-glycans is often overlooked and underexplored. This is
partially due to the lack of a universal enzyme for the release of O-glycans from the protein backbone. Furthermore, the traditional
chemical releasing method suffers from severe side reactions and involves
tedious sample preparation procedures. Here, a multiplexed isobaric
labeling method enabled by N,N-dimethyl
leucine containing pyrazolone analogue (DiLeuPMP) is introduced. This
method combines the release and labeling of O-glycans
in a one-pot reaction and achieves accurate MS2-based relative
quantification with the ability to process four samples at a time.
The method has been applied to core-1 O-glycan standard
and three glycoproteins first, and the results demonstrated its validity.
Following this proof-of-principle demonstration, we analyzed more
complex biological specimen using human serum samples. Overall, this
method provides an effective and reliable approach for the profiling
and high-throughput quantitative analysis of O-glycans
in complex samples.