Bovine milk-derived exosomes (BMDEs) have potential applications in the pharmaceutical industry as drug delivery carriers. A comprehensive analysis of protein glycosylation in exosomes is necessary to elucidate the process of targeted delivery. In this work, free oligosaccharides (FOSs), O-glycans, and N-glycans in BMDEs and whey were first analyzed through multiple derivation strategies. In summary, 13 FOSs, 44 O-glycans, and 94 N-glycans were identified in bovine milk. To analyze site-specific glycosylation of glycoproteins, a one-step method was used to enrich and characterize intact glycopeptides. A total of 1359 proteins including 114 glycoproteins were identified and most of these were located in the exosomes. Approximately 95 glycopeptides were initially discovered and 5 predicted glycosites were confirmed in BMDEs. Collectively, these findings revealed the characterization and distribution of glycans and glycoproteins in BMDEs, providing insight into the potential applications of BMDEs in drug delivery and food science.

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Castration-resistance of prostate cancer is one of the most challenging clinical problems. In the present study, we have performed proteomics and glycomics using LNCaP model. Growth differentiation factor-15 (GDF15) level is increased in androgen receptor (AR) inhibitor-resistant cells and the inhibitory effect of GDF15 on epithelial growth factor receptor (EGFR) pathway is relieved by GDF15 N70 glycosylation. Interference of GDF15 (siRNA or N70Q dominant negative) or EGFR pathway (inhibitor or siRNA for EGFR, SRC or ERK) decreases the resistant-cell survival in culture and tumor growth in mice. Our study reveals a novel regulatory mechanism of prostate cancer AR inhibitor resistance, raises the possibility of AR/SRC dual-targeting of castration-resistance of prostate cancer, and lays foundation for the future development of selective inhibitors of GDF15 glycosylation.

Background Breast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. The prognosis and survival of the two subtypes were significantly different even with specific molecular subgroup. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in same molecular subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis. Methods Cancer tissues and noncancerous adjacent tissues (NATs) with the luminal subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differentially expressed proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, bioinformatics analysis was performed to explore the difference between IDC and ILC subtypes, including the difference in protein expression levels and the degree of phosphorylation. Meanwhile, Kinase-Substrate Enrichment Analysis (KSEA) revealed the activation difference of kinases and their substrates between IDC and ILC. Results A total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from breast cancer tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. From the quantitative analysis of 1259 proteins and 560 phosphoproteins with high patient coverage, Histone H1.10, Complement C4-B and Crk-like protein were found to be significantly differentially expressed in the two subtype tissues from the proteomics analysis. Moreover, the differences in protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin and their phosphorylation clearly distinguished IDC from ILC. In addition, differentially expressed proteins and differentially expressed phosphoproteins in IDC were primarily related to energy metabolism and MAPK pathway, while ILC subtypes were more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes from IDC were primarily significantly activated in the CMGC (Cyclin-dependent kinases, Mitogen-activated protein kinases, Glycogen synthase kinases and CDK-like kinases) groups. Conclusions Our research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.

BackgroundBreast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in luminal A subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis.MethodsCancer tissues and noncancerous adjacent tissues (NATs) with the luminal A subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differential proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, the difference in protein expression and its phosphorylation between IDC and ILC subtypes were explored. Meanwhile, the activation of kinases and their substrates was also revealed by Kinase-Substrate Enrichment Analysis (KSEA).ResultsIn the luminal A breast cancer, a total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from 10 paired tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. Histone H1.10 was significantly increased in IDC but decreased in ILC, Conversely, complement C4-B and Crk-like protein were significantly decreased in IDC but increased in ILC. Moreover, the increased protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin but reduce of their phosphorylation could clearly distinguish IDC from ILC. In addition, IDC was primarily related to energy metabolism and MAPK pathway, while ILC was more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes in IDC were primarily significantly activated in the CMGC groups.ConclusionsOur research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.