In-Gel Nonspecific Proteolysis
for Elucidating Glycoproteins:
A Method for Targeted Protein-Specific Glycosylation Analysis in Complex
Protein Mixtures
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Abstract
Determining protein-specific glycosylation in protein
mixtures
remains a difficult task. A common approach is to use gel electrophoresis
to isolate the protein followed by glycan release from the identified
band. However, gel bands are often composed of several proteins. Hence,
release of glycans from specific bands often yields products not from
a single protein but a composite. As an alternative, we present an
approach whereby glycans are released with peptide tags allowing verification
of glycans bound to specific proteins. We term the process in-gel
nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG
combines rapid gel separation of a protein mixture with in-gel nonspecific
proteolysis of protein bands followed by tandem mass spectrometry
(MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel
digestion is shown for the first time with nonspecific and broad specific
proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin.
Tandem MS analysis of the resulting glycopeptides separated on a porous
graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography
coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF
MS). In this study, rapid and automated glycopeptide assignment was
achieved via an in-house software (Glycopeptide Finder) based on a
combination of accurate mass measurement, tandem MS data, and predetermined
protein identification (obtained via routine shotgun analysis). INPEG
is here initially validated for O-glycosylation (κ casein) and
N-glycosylation (ribonuclease B). Applications of INPEG were further
demonstrated for the rapid determination of detailed site-specific
glycosylation of lactoferrin and transferrin following gel separation
and INPEG analysis on crude bovine milk and human serum, respectively