pr4000398_si_004.pdf (143.57 kB)
Quantitative Analysis of Glycated Proteins
journal contribution
posted on 2014-02-07, 00:00 authored by Feliciano Priego-Capote, María Ramírez-Boo, Francesco Finamore, Florent Gluck, Jean-Charles SanchezThe proposed protocol presents a
comprehensive approach for large-scale qualitative and quantitative
analysis of glycated proteins (GP) in complex biological samples including
biological fluids and cell lysates such as plasma and red blood cells.
The method, named glycation isotopic labeling (GIL), is based on the
differential labeling of proteins with isotopic [13C6]-glucose, which supports quantitation of the resulting glycated
peptides after enzymatic digestion with endoproteinase Glu-C. The
key principle of the GIL approach is the detection of doublet signals
for each glycated peptide in MS precursor scanning (glycated peptide
with in vivo [12C6]- and in vitro [13C6]-glucose). The mass
shift of the doublet signals is +6, +3 or +2 Da depending on the peptide
charge state and the number of glycation sites. The intensity ratio
between doublet signals generates quantitative information of glycated
proteins that can be related to the glycemic state of the studied
samples. Tandem mass spectrometry with high-energy collisional dissociation
(HCD–MS2) and data-dependent methods with collision-induced
dissociation (CID–MS3 neutral loss scan) are used for qualitative
analysis.