关键词: 富集, 二硫化钼, 4-巯基苯硼酸, 外泌体, N-糖基化肽段

Abstract:

Protein N-glycosylation plays a crucial role in the folding, transportation, and localization of proteins, and participates in many important biological processes such as receptor activation and signal transduction. An increasing number of studies have shown that abnormal protein glycosylation is closely related to various diseases. Therefore, N-glycosylated proteins are potential candidates for become new biomarkers or drug targets. The current research strategy for N-glycosylated proteins is to first digest the protease into peptides and then identify them by mass spectrometry. In sample preparation processing, enrichment and separation of N-glycopeptides have become the vital step for glycoproteomic analysis. However, because of their low abundance and poor ionization, mass spectrometric identification of N-glycopeptides in complex samples remains a challenging task. Therefore, the development of a selective enrichment strategy for N-glycopeptides from complex biological samples is necessary. In this work, new functional nanomaterials were prepared through a simple, convenient, and efficient two-step "Au-S" reaction. In short, the "Au-S" bond was first used to load nano-gold wires on the surface of molybdenum disulfide (MoS₂), and then to connect these wires to 4-mercaptophenylboronic acid (4-MPB). The resulting nanomaterial named MoS₂/Au/4-MPB was successfully prepared by serial functionalization of ultra-thin two-dimensional MoS₂, nano-gold wires and 4-MPB for the efficient enrichment of N-glycopeptides. The layered structure of molybdenum disulfide nanomaterials can provide numerous modifiable sites for the reaction, allowing for convenient modification of the nano-gold wires. The functional group 4-MPB has high affinity for N-glycopeptides, and it can selectively enrich them in biological samples. In order to evaluate the N-glycopeptide enrichment performance of MoS₂/Au/4-MPB, standard proteins human immunoglobulin G (IgG) and bovine serum albumin (BSA) trypsin digests were used. Thus, facilitated interactions with N-glycopeptides for boric acid-based retention could be expected. Low femtomolar detection sensitivity, 1:1000 enrichment selectivity, and 100 μg/mg loading capacity were achieved for N-glycopeptide enrichment. In the application of this material to biological samples, exosomes were chosen as the research objects. On the one hand, exosomes serve as a new class of carriers for intercellular communication and drug delivery. Almost all types of cells can secrete exosomes. On the other hand, urine is another potential source for liquid biopsy samples in addition to blood. Obtaining urine samples is a less invasive process, and urine samples are easier to handle and save as compared to blood samples. Exosomes can also be detected in urine. Cell-specific components such as proteins, lipids, and nucleic acids can provide diverse information, which is of great significance in disease diagnosis and treatment. However, there are only a few studies on urinary exosomes N-glycoprotein group. In this research, N-glycoproteins were successfully identified in urine exosomes. In three replicate experiments, 536, 515 and 487 N-glycosylated peptides were identified from 279, 270 and 279 N-glycoproteins, respectively. After de-duplication of the three sets of data, a total of 768 N-glycopeptides corresponding to 377 N-glycoproteins were identified. This result indicates that the novel nanocomposite has good enrichment selectivity and sensitivity for N-glycopeptides enrichment in complex biological samples, and this method provides a new method for glycoproteomics research.

Key words: enrichment, molybdenum disulfide, 4-mercaptophenylboronic acid, exosomes, N-glycopeptides