Journal: Molecular & Cellular Proteomics

Article Title: Quantification of the N-glycosylated Secretome by Super-SILAC During Breast Cancer Progression and in Human Blood Samples

doi: 10.1074/mcp.m112.023614

Figure Lengend Snippet: FIG. 1. Overview of the experimental workflow. A, The secretome was collected as conditioned medium from cell lines in culture. HCC1143, HCC1937 and HCC2218 were also cultured under SILAC conditions to generate a super-SILAC mix as an internal standard. B, N-glycosylated peptides were enriched using N-glyco FASP. Proteins were first digested to peptides and N-glycosylated peptides were then captured using lectins. Finally, N-glycosylated peptides were released by deglycosylation using PNGase F and the deglycosylated peptides were analyzed by LC-MS.

Article Snippet: HCC202, and HCC2218 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA); HCC1143, HCC1937, and HCC1599 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ); MCF-10a and MDA-MB-453 cells were kindly provided by Axel Ulrich (Max-Planck Institute of Biochemistry, Martinsried, Germany).

Techniques: Cell Culture, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular & Cellular Proteomics

Article Title: Quantification of the N-glycosylated Secretome by Super-SILAC During Breast Cancer Progression and in Human Blood Samples

doi: 10.1074/mcp.m112.023614

Figure Lengend Snippet: FIG. 3. Effect of normalization using the super-SILAC internal standard. A, Correlation of intensities of light N-glycosylated peptides intensities between different replicates and cell lines (light channel of SILAC measurements only). Samples are ordered horizontally and vertically according to replicates and cell line (from HMEpC1 until MDA-MB-453). B, Correlation of intensities of heavy isotope labeled N-glycosylated peptides (super-SILAC internal standard) intensities between different replicates and cell lines. C, Correlation of N-glycosylated peptide ratios between different replicates and cell lines after normalization using the super-SILAC internal standard. Correlations between replicates (“green blocks” near the diagonal) and cell lines from the same cancer stages are increased, whereas differences with other cell lines are augmented. D, Correlation between replicates from each cell line at the N-glycosylated peptide level (from panel A and C) and protein level (based on nonglycosylated and glycosylated peptides) before and after normalization using the super-SILAC internal standard. Pearson’s r was calculated between all replicates from each cell line. Two aberrant measurements (one from HMEpC2 and one from HCC1937) were omitted.

Article Snippet: HCC202, and HCC2218 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA); HCC1143, HCC1937, and HCC1599 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ); MCF-10a and MDA-MB-453 cells were kindly provided by Axel Ulrich (Max-Planck Institute of Biochemistry, Martinsried, Germany).

Techniques: Multiplex sample analysis, Labeling