Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.

N-linked glycosylation is prevalent in proteins destined for extracellular environments; nearly all secreted proteins are glycosylated. However, with respect to their glycosylation sites, little attention has been paid. Here, we report the analysis of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells. For the enrichment of glycopeptides, capture methods with hydrophilic affinity (HA) and hydrazide chemistry (HC) were used complementarily. With the use of both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% vs 51.3%); however, based on the number of glycosites identified, HA outweighs HC (265 vs 159). Furthermore, unavoidable contaminants such as actin and bovine serum albumin which are not N-glycosylated could be easily depleted by using this glycoproteomic strategy. As a consequence, more low-abundance and genuinely secreted proteins were identified. Among the glycoproteins identified, alpha-fetoprotein, CD44 and laminin have been reported to be implicated in HCC and its metastasis.

Metastasis is a major clinical obstacle responsible for the high mortality and poor prognosis of gastric cancer (GC). MicroRNAs (miRNAs) are critical mediators of metastasis that act by modulating their target genes. In this study, we found that miR-143 and miR-145 act via a common target gene, MYO6, to regulate the epithelial–mesenchymal transition (EMT) and inhibit metastasis. We determined that miR-143 and miR-145 were downregulated in GC, and the ectopic expression of miR-143 and/or miR-145 inhibited GC cell migration and metastasis. Furthermore, MYO6 was identified as a direct common target of miR-143 and miR-145 and was elevated in GC. Silencing of MYO6 resulted in a metastasis-suppressive activity similar to that of miR-143 and miR-145, while restoring MYO6 attenuated the anti-metastatic or anti-EMT effects caused by miR-143 and miR-145. Clinically, an inverse correlation was observed between miR-143/145 levels and MYO6 levels in GC tissues, and either miR-143/145 downregulation or MYO6 upregulation was associated with more malignant phenotypes in patients with GC. In conclusion, miR-143 and miR-145 suppress GC cell migration and metastasis by inhibiting MYO6 expression and the EMT, which provides a novel mechanism and promising therapeutic target for the treatment of GC metastasis.