TY - JOUR

T1 - HCD Fragmentation of Glycated Peptides

AU - Keilhauer, Eva C

AU - Geyer, Philipp E

AU - Mann, Matthias

PY - 2016/8/5

Y1 - 2016/8/5

N2 - 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.

AB - 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.

KW - Arginine

KW - Diabetes Mellitus

KW - Glycation End Products, Advanced

KW - Glycopeptides

KW - Glycosylation

KW - HeLa Cells

KW - Humans

KW - Lysine

KW - Mass Spectrometry

KW - Peptide Fragments

KW - Proteome

KW - Proteomics

KW - Journal Article

U2 - 10.1021/acs.jproteome.6b00464

DO - 10.1021/acs.jproteome.6b00464

M3 - Journal article

C2 - 27425404

VL - 15

SP - 2881

EP - 2890

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 8

ER -