Abstract
N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples.
Highlights
Glycosylation is one of the most prominent post-translational modification methods for proteins (Stadlmann et al, 2017; Huang et al, 2019)
The Oleic acid (OA)-coated Fe3O4 nanoparticles were synthesized according to the method, and the initiators were immobilized on the surface of Fe3O4 nanoparticles through ligand exchange reaction
thermoresponsive magnetic fluid (TMF) was characterized by dynamic light scattering (DLS), Fourier transform-infrared (FT-IR) spectroscopy, Vibrating sample magnetometry (VSM), and thermogravimetric analysis (TGA) to confirm the successful growth of PNIPAM-b-PUnAl copolymer chains on the surface of the magnetic nanoparticles and determine the content of the grafted copolymer chains
Summary
Glycosylation is one of the most prominent post-translational modification methods for proteins (Stadlmann et al, 2017; Huang et al, 2019). The N-glycans biosynthesis process is very sensitive to the physiological and pathological states in cells (Mereiter et al, 2019; Dong et al, 2020), and glycoproteins are a main type of current therapeutic targets and disease biomarkers (Pan et al, 2020; Zhao et al, 2020). As a reflection of body changes, is considered as an ideal source for biomarker discovery (Wu and Gao, 2015). It can be obtained in a non-invasive manner with a relatively narrower protein dynamic range and much less interference by high-abundance proteins compared with that of plasma/serum (Zhao et al, 2017). Rapid and in-depth analysis of Nglycoproteome from a large cohort of urine samples is highly desirable for a clinical study
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
