HCD Fragmentation of Glycated Peptides

Protein glycation
is a concentration-dependent nonenzymatic reaction
of reducing sugars with amine groups of proteins to form early as
well as advanced glycation (end-) products (AGEs). Glycation is a
highly disease-relevant modification but is typically only studied
on a few blood proteins. To complement our blood proteomics studies
in diabetics, we here investigate protein glycation by higher energy
collisional dissociation (HCD) fragmentation on Orbitrap mass spectrometers.
We established parameters to most efficiently fragment and identify
early glycation products on in vitro glycated model proteins. Retaining
standard collision energies does not degrade performance if the most
dominant neutral loss of H6O3 is included into
the database search strategy. Glycation analysis of the entire HeLa
proteome revealed an unexpected intracellular preponderance for arginine
over lysine modification in early and advanced glycation (end-) products.
Single-run analysis from 1 μL of undepleted and unenriched blood
plasma identified 101 early glycation sites as well as numerous AGE
sites on diverse plasma proteins. We conclude that HCD fragmentation
is well-suited for analyzing glycated peptides and that the diabetic
status of patients can be directly diagnosed from single-run plasma
proteomics measurements.