Protein glycosylation
is a prevalent post-translational modification
that mediates a variety of cellular processes. For membrane proteins,
glycosylation at their terminal motif is usually more functional.
Among the various glycosylation types found in membrane proteins,
O-glycosylation is the most common and is closely correlated with
a variety of cancer types, including breast cancer. Slightly aberrant
expression of certain O-glycans can significantly
affect cancer progression, especially at the cancer-related membrane
protein level. To collect biological information on protein-specific
glycosylation and further explore clinical applications, quantitative
detection of glycosylation is essential. However, few assays have
been reported for the in situ detection of protein-specific
glycosylation to date. Herein, we developed a dual-probe approach
for mass spectrometric quantification of protein-specific glycosylation
using the terminal galactose/N-acetylgalactosamine
(Gal/GalNAc) of MUC1 as a model. The dual-probe (i.e., protein probe
and glycan probe) system was first designed and built. The protein
probe contained an aptamer for MUC1 protein recognition and a capture
DNA sequence. Correspondingly, the glycan probe had a DNA sequence
complementary to that of the capture DNA, a substrate peptide containing
a reporter peptide, and a tryptic cleavage site, and could be covalently
linked with the terminal Gal/GalNAc. Exonuclease III enabled recycling
of the hybridization–dehybridization process in a restricted
space. Finally, the reporter peptide was tryptically released and
quantified by liquid chromatography–tandem mass spectrometry
(LC–MS/MS). The mass response of the reporter peptide represented
the amount of MUC1-specific terminal Gal/GalNAc. This dual-probe approach
was applied for in situ detection of MUC1-specific
terminal Gal/GalNAc in three human breast cancer cell lines and 32
pairs of matched breast cancer tissue samples. The relationship between
MUC1-specific terminal Gal/GalNAc expression and breast cancer diagnosis/prognosis
was also assessed.