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Site-specific N-linked glycosylation analysis of human carcinoembryonic antigen

ABSTRACT: CE-MS/MS analysis of a pure carcinoembryonic antigen obtained from patient tissue samples. Enzymes used for digestion: trypsin, Glu-C, pronase. CE separation voltage 20 kV, normal polarity. CE-MS/MS parameters: ESI positive mode, glass capillary voltage at 1200 V, drying gas temperature at 150C, drying gas flow rate at 1.2 L/min, nebulizer gas pressure at 0.2 bar, quadrupole ion energy at 3.0 eV and collision cell energy at 7.0 eV. MS data were acquired between m/z 200 and 2000 with a spectral acquisition rate of 1 Hz. MS/MS spectra were acquired in a data dependent mode with an absolute threshold of 4548 counts and active exclusion. D1 - CEA1 trypsin, D2 - CEA2 trypsin, D3 - CEA3 trypsin, D4 - CEA1 Glu-C, D5 CEA2 Glu-C, D6 - CEA3 Glu-C, D7 - CEA1 pronase, D8 - CEA2 pronase, D9 - CEA3 pronase

INSTRUMENT(S): Impact HD UHR-QqTOF-MS

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Guinevere Lageveen-Kammeijer

PROVIDER: MSV000086774 | MassIVE | Thu Jan 28 00:22:00 GMT 2021

REPOSITORIES: MassIVE

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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

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Site-specific N-linked glycosylation analysis of human carcinoembryonic antigen

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