Cells secrete a large number of proteins to communicate with their surroundings. 
Furthermore, plasma membrane proteins and intracellular proteins can be released 
into the extracellular space by regulated or non-regulated processes. Here, we 
profiled the supernatant of 11 cell lines that are representative of different 
stages of breast cancer development by specifically capturing N-glycosylated 
peptides using the N-glyco FASP technology. For accurate quantification we 
developed a super-SILAC mix from several labeled breast cancer cell lines and 
used it as an internal standard for all samples. In total, 1398 unique 
N-glycosylation sites were identified and quantified. Enriching for 
N-glycosylated peptides focused the analysis on classically secreted and 
membrane proteins. N-glycosylated secretome profiles correctly clustered the 
different cell lines to their respective cancer stage, suggesting that 
biologically relevant differences were detected. Five different profiles of 
glycoprotein dynamics during cancer development were detected, and they 
contained several proteins with known roles in breast cancer. We then used the 
super-SILAC mix in plasma, which led to the quantification of a large number of 
the previously identified N-glycopeptides in this important body fluid. The 
combination of quantifying the secretome of cancer cell lines and of human 
plasma with a super-SILAC approach appears to be a promising new approach for 
finding markers of disease.