O-Glycome Profiling of Breast Cancer Cell Lines to Understand Breast Cancer Brain Metastasis.

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O-glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O-glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O-glycan compositions. We observed significant upregulation in the expression of HexNAc1Hex1NeuAc2 and HexNAc2Hex3, whereas the expression of HexNAc1Hex1NeuAc1 was downregulated in MDA-MB-231BR compared to other cell lines. In our isomeric analysis, we observed notable alterations in the isomeric forms of the O-glycan structure HexNAc1Hex1NeuAc1 in a comparison of different cell lines. Our analysis of O-glycans and their isomers in cancer cells demonstrated that changes in their distribution can be related to the metastatic process. We believe that our investigation will contribute to an enhanced comprehension of the significance of O-glycans and their isomers in BCBM.

15N metabolic labeling-TMT multiplexing approach to facilitate the quantitation of glycopeptides derived from cell lines.

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles.

O-Glycoproteomics Sample Preparation and Analysis Using NanoHPLC and Tandem MS.

Glycosylation refers to the biological processes that covalently attach carbohydrates to the peptide backbone after the synthesis of proteins. As one of the most common post-translational modifications (PTMs), glycosylation can greatly affect proteins' features and functions. Moreover, aberrant glycosylation has been linked to various diseases. There are two major types of glycosylation, known as N-linked and O-linked glycosylation. Here, we focus on O-linked glycosylation and thoroughly describe a bottom-up strategy to perform O-linked glycoproteomics studies. The experimental section involves enzymatic digestions using trypsin and O-glycoprotease at 37 °C. The prepared samples containing O-glycopeptides are analyzed using nanoHPLC coupled with tandem mass spectrometry (MS) for accurate identification and quantification.

LC-MS/MS Quantitation of HILIC-Enriched N-glycopeptides Derived from Low-Abundance Serum Glycoproteins in Patients with Narcolepsy Type 1.

Glycoproteomic analysis is always challenging because of low abundance and complex site-specific heterogeneity. Glycoproteins are involved in various biological processes such as cell signaling, adhesion, and cell-cell communication and may serve as potential biomarkers when analyzing different diseases. Here, we investigate glycoproteins in narcolepsy type 1 (NT1) disease, a form of narcolepsy characterized by cataplexy-the sudden onset of muscle paralysis that is typically triggered by intense emotions. In this study, 27 human blood serum samples were analyzed, 16 from NT1 patients and 11 from healthy individuals serving as controls. We quantified hydrophilic interaction liquid chromatography (HILIC)-enriched glycopeptides from low-abundance serum samples of controls and NT1 patients via LC-MS/MS. Twenty-eight unique N-glycopeptides showed significant changes between the two studied groups. The sialylated N-glycopeptide structures LPTQNITFQTESSVAEQEAEFQSPK HexNAc6, Hex3, Neu5Ac2 (derived from the ITIH4 protein) and the structure IVLDPSGSMNIYLVLDGSDSIGASNFTGAK HexNAc5, Hex4, Fuc1 (derived from the CFB protein), with p values of 0.008 and 0.01, respectively, were elevated in NT1 samples compared with controls. In addition, the N-glycopeptide protein sources Ceruloplasmin, Complement factor B, and ITH4 were observed to play an important role in the complement activation and acute-phase response signaling pathways. This may explain the possible association between the biomarkers and pathophysiological effects.