Figure 5

Liquid chromatography–mass spectrometry (LC–MS)-based proteomic analyses reveal expression of additional cell surface markers in CD133⁺ colon tumour spheroid cells. (A) Mass spectrometric intensity plots of glycopeptides. Peptide intensities of replicates for both spheroid (left) and primary tumour cell (middle) samples show tight correlation, indicating a similar expression level within the sample. The intensity profile changed dramatically from spheroid to primary cell populations (right), indicating that glycoprotein expression profiles are different in the two populations. Among those differentially expressed proteins, overexpressed proteins (peptides) in spheroid sample (>4-fold) were further sequenced for identification. (B) Intensity comparison of glycopeptides from CD133 (LPIQDILSAFSVYVNNTESYIHR) and CD44 (AFNSTLPTMAQMEK) detected in primary vs cultured spheroid cell samples. Intensities of these peptides were significantly increased in tumour spheroid cells (11-fold and 45-fold, respectively) as shown in extracted ion current (XIC) chromatograms (upper panel) and corresponding mass spectra (lower panel).