2.1 Cell culture and protein extraction
BC cell lines for MDA-MB-231, MDA-MB-453, MDA-MB-157, and BT549 cells were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) in a humidified atmosphere with 5% CO2 at 37 ℃. For protein extraction, the cells were washed with PBS and then incubated with denaturing buffer supplemented with 8 M urea and 1 M ammonium bicarbonate. To eliminate biological variations, cellular proteins were extracted from three different biological replicates of each condition and merged into a single sample for additional sample processing. The protein solution was then reduced with 5 mM dithiothreitol (DTT) and the reaction was agitated at 37°C for 1 h to fully open the disulfide links in the protein. The reaction was then carried out at room temperature with the addition of iodoacetamide (IAM) at a final concentration of 15 mM, shielded from light, and shaken for 30 minutes. Finally, the reaction was ended by adding DTT at a final concentration of 2.5 mM, and the leftover IAM was reacted by incubating for 10 minutes at room temperature. The protein solution was diluted to a concentration of 1 M urea with HPLC grade purified water, sequencing grade trypsin (Promega, USA) was added in a 50:1 ratio to the amount of protein, and the reaction was carried out overnight at 37 ℃ with gentle shaking. A 50% trifluoroacetic acid (TFA) solution was added to each sample, vortexed, and agitated, pH paper was tested to verify pH < 2, and samples were centrifuged for 15 minutes at 13,000 rpm/min to obtain the supernatant. The peptides in the supernatant were desalted using the C18 column (Waters, USA).
2.2 Enrichment of glycopeptides in BC cells
The intact glycopeptides were enriched using hydrophilic interaction chromatography (HILIC) micro-column (Agela Technologies, Tianjin, China). Briefly, the activation column was rinsed with 80% acetonitrile (ACN)/1% TFA. Subsequently, the concentrated digestion mixture, dissolved in 50 µl of 80% ACN/1% TFA solution, was up-sampled on the HLIC column. Then desalting was performed with 100 µl of 80% ACN/1% TFA solution for 3 times. Finally, the glycopeptides were collected by elution with 100 µL of 5% ACN/1% TFA solution. The eluted solution was spun dry by freeze-drying centrifuge and stored at -20°C for use.
2.3 LC-MS/MS analysis
The enriched glycopeptides were analyzed by Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific, Germany). The following mass spectrometry parameters were used for proteome analysis: 2.4 kV was set as the spray voltage. Primary mass spectrometry (MS1) collected spectra from 350–1800 m/z at 120 K resolution with a maximum ion injection of 4×105, selecting the valence state of the parent ions from 2- to 7-valence; secondary mass spectrometry (MS2) fragmentation was by high-energy collision dissociation (HCD) with reaction times of 1 ms, collision energies of 20% and 30%, and a selective isolation width of 1.6 Da for the parent ion, with spectra collected from 150–2000 m/z at 50 K resolution, a maximum ion injection time of 105 ms, and a dynamic exclusion time of 25 s for the selection of the parent ion. Glycoproteomics mass spectrometry parameters were detailed as follows: the MS1 collected spectra from 350–2000 m/z at 120K resolution with a maximum ion injection of 4×105, selecting the parent ions with valence states from 2- to 7-valence; the secondary mass spectrometry (MS2) fragmentation was by HCD, with a reaction time of 1 ms, collision energies of 20 and 30%, and a selective isolation width of the parent ions of 1.6 Da at a Spectra were collected from 110–3000 m/z at 50 K resolution, the maximum ion injection time was 105 ms, and a dynamic exclusion time of 5 s was used to select the parent ion.
2.4 Identification and visualization of intact glycopeptides by StrucGP
The identification of intact glycopeptides was carried out by using StrucGP [25]. In short, all MS data were converted into “mzML” format by the Trans-Proteomic Pipeline (TPP, v.5.0.0, ). The intact glycopeptide analyses were performed using the built-in glycan branch structure database from StrucGP and the human protein databases from Uniprot (https://www.uniprot.org/). Finally, the GlycoVisual tool was utilized for automatically displaying the corresponding MS/MS spectra from the StrucGP results.
2.5 Bioinformatics analysis
Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of Fucose (F)-modified glycoproteins and unique glycoproteins in MDA-MB-231 and MDA-MB-453 cells were performed by Metascape [26]. And, the STRING 11.0 (http://string-db.org) database was used to explore the protein-protein Interaction (PPI) network of F-modified glycoproteins and unique glycoproteins. Moreover, the expression of the hub glycoproteins in BC tissues and normal tissues was analyzed by the online software UALCAN (http://ualcan.path.uab.edu/analysis.html). In our study, we investigated the difference in hub glycoprotein expression from distinguished perspectives of TNM stage and sample types. In addition, the relationship between the glycoproteins and the prognosis of BC was assessed using the Kaplan-Meier plotter database (http://kmplot.com) [27].
2.6 Cell transfection
The overexpression plasmids of TXM3 (oe-TXM3#1 and oe-TXM3#2) and normal controls (oe-NC) were provided by GenePharma (Shanghai, China). For cell transfection, lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA) was used to transfect TXM3 overexpression plasmid and its control vector into MDA-MB-231 and MDA-MB-453 cells according to the manufacturer’s procedures. After transfection for 24h, the transfection efficiency was estimated via Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) and Western Blot assays.
2.7 CCK-8 assay
Cell viability was measured by a Cell Counting Kit-8 (CCK-8, Beyotime, China). Briefly, MDA-MB-231 and MDA-MB-453 cells post-transfection were cultured in 96-well plates at a density of 3×103 cells/well for 24h, 48h, 72h, 96h, and 120h, respectively. Then, 10 µL of CCK-8 solution was added to each well for 1h incubation at 37°C. After that, the absorbance at 450 nm was assessed by a microplate reader (CMaxPlus, Molecular Devices).
2.8 Colony formation assay
Cell colony formation assay was adopted to analyze the cell proliferation of cells. MDA-MB-231 and MDA-MB-453 cells were seeded into 6-well plates at a density of 1×103 cells/well. After the cells were transfected and/or exposed to 2 µmol/L of Notch1 agonist (NSC 22423, Sigma, USA), the culture was then continued for 14 days to form cell colonies. After 2 weeks, MDA-MB-231 and MDA-MB-453 cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. The cell clones were counted under a microscope (Olympus, Tokyo, Japan).
2.9 Flow cytometry assay
Cell apoptosis was measured using the BD 556547 Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD Pharmingen, USA) according to the manufacturer's instructions. In short, the transfected MDA-MB-231 and MDA-MB-453 cells were incubated with 10 µL of Annexin V-FITC and 5 µL of PI for 15 minutes at room temperature in the dark. Next, the cells were loaded onto the NovoCyte flow cytometer (Agilent, USA). The flow cytometry data were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).
2.10 Transwell migration assay
Transfected MDA-MB-231 and MDA-MB-453 cells were resuspended and adjusted to 1 × 105 cells/ml in serum-free medium, and 100 µL cells were transferred into the upper layer of the Transwell chamber. The lower layer of chambers was filled with 600 µL complete medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). After cultivation for 24 h, the cells in the lower chambers were fixed with 70% ethanol for 15 minutes and stained with 4% crystal violet solution (Sigma-Aldrich). Subsequently, the number of cells was counted under a fluorescence microscope.
2.11 qRT-PCR assay
Total RNA was extracted from BC cell lines using TRIzol reagent (Invitrogen, USA). Then, the reverse transcription was performed according to the PrimeScript RT Reagent Kit (Takara, Dalian, China). Next, the qPCR was performed using SYBR Green qPCR Super Mix (Takara) on the LightCycler® 96 Detection System (Roche, Shanghai, China). The primers were as follows: TMX3: Forward, 5’- ATCTGGGGCTCTAATTCGGC-3’; Reverse, 5’‐ AGTGTCACATACTCAGGAACCA-3’. β-actin: Forward, 5’‐ GATGACCCAGATCATGTTTGAG-3’; Reverse, 5’‐ TAATGTCACGCACGATTTCC-3’. The relative expression of TMX3 was analyzed by the 2−ΔΔCt method. β-actin is used as the internal reference for TMX3.
2.12 Western blot assay
Total protein from the BC cell lines and the transfected MDA-MB-231 and MDA-MB-453 cells was extracted using RIPA lysis buffer (Beyotime, China). After quantification of total protein using the BCA Kit (Beyotime), a total of 30 µg protein sample was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat dried milk for 2 h, the membranes were incubated with primary antibodies against anti-TMX3 (1:1000, Proteintech Group, Inc, USA), anti-Bax (1:1000, Affinity Biosciences, Jiangsu, China), anti-Bcl-2 (1:1000, Affinity), anti-Cleaved caspase-3 (1:1000, Abcam), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-N-cadherin (1:1000, Affinity), anti-Jagged1 (JAG1, 1:1000, BEIJING BIOSYNTHESIS BIOTECHNOLOGY CO., LTD. China), anti-NOTCH1 (1:1000, Abcam), anti-NOTCH3 (1:1000, Affinity), and anti-β-actin (1:5000, Proteintech) overnight at 4°C. After that, the membranes were treated with HRP-labeled secondary antibody (1:5000, Cell Signaling Technology) for 1h at room temperature. The protein bands were visualized through the enhanced chemiluminescence kit (Beyotime) and analyzed using ImageJ software.
2.13 Xenograft nude mice assay
Male Five-week-old immunodeficient BABL/c nude mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (SCXK (HU) 2017-0005, Shanghai, China) and placed in specific pathogen-free conditions with 50% humidity, a 12 h light/dark cycle and constant temperature of 22 ± 2°C. After an additional week of adaptation, HCT15 cells (~ 1×106) in 100 µL were subcutaneously injected into the right flank of the mouse. After 7 days, the tumor volume was measured every 3 days, and the tumor volume was calculated as follows: V (mm3) = L(length) × W2(width)/2. At the end of the experiment, the mice were sacrificed under excessive inhalation of carbon dioxide, and the tumor tissues were collected, weighed, photographed, and used for immunohistochemical analysis. All experiments and animal care were approved by the Ethics Committee of Zhejiang Eyong Pharmaceutical Research and Development Center (No. ZJEY-20230420-05).
2.14 Immunohistochemical assay
The paraformaldehyde-fixed and paraffin-embedded tumor tissues were made into continuous 4µm pathology sections. After dewaxing, dehydrating, antigen retrieving, and endogenous peroxidase blocking for the sections, the sections were incubated with the indicated primary antibodies for 2h, and then incubated with horseradish peroxidase-linked secondary antibody. After that, visualization was performed by using a 3,3′-diaminobenzidine (DAB, Fuzhou Maixin Biotech. Co., Ltd., China) staining kit followed by counterstaining with hematoxylin. Finally, the images were captured and quantified with Image Pro Plus 6.0 software (Media Cybernetics, Inc.).
2.15 Statistical analysis
All data were presented as mean ± SD. SPSS software 21.0 was used to perform the statistical analyses. Student’s t-test was employed for the comparisons between the two groups, and a P-value < 0.05 was considered statistically significant.