Profiling of Methylglyoxal Blood Metabolism and Advanced Glycation End-Product Proteome Using a Chemical Probe

Abstract

Methylglyoxal (MG) is quantitatively the most important precursor to advanced glycation end-products (AGEs), and evidence is accumulating that it is also a causally linked to diabetes and aging related diseases. Living systems primarily reside on the glyoxalase system to detoxify MG into benign D-lactate. The flux to either glycation or detoxification, accordingly, is a key parameter for how well a system handles the ubiquitous glyoxal burden. Furthermore, insight into proteins and in particular their individual modification sites are central to understanding the involvement of MG and AGE in diabetes and aging related diseases. Here, we present a simple method to simultaneously monitor the flux of MG both to D-lactate and to protein AGE formation in a biological sample by employing an alkyne-labeled methylglyoxal probe. We apply the method to blood and plasma to demonstrate the impact of blood cell glyoxalase activity on plasma protein AGE formation. We move on to isolate proteins modified by the MG probe and accordingly can present the first general inventory of more than 100 proteins and 300 binding sites of the methylglyoxal probe on plasma as well as erythrocytic proteins. Some of the data could be validated against a number of in vivo and in vitro targets for advanced glycation previously known from the literature; the majority of proteins and specific sites however were previously unknown and may guide future research into MG and AGE to elucidate how these are functionally linked to diabetic disease and aging.

STEFs: Activated Vinylogous Protein‐Reactive Electrophiles

“…Next, lysates were pretreated with iodoacetamide before exposure to STEFs ( Figure 3B). As expected, this completely blocked IA-alkyne labelling but, interestingly,a lso strongly reduced labelling with STEFs, especially with 9.This data further supports arole for cysteine residues as direct targets and, as suggested by the previous experiments,a sintermediary conjugation sites towards irreversible amine conjugations.T op robe these questions,w e sought the specific protein targets of 9.W ep erformed an affinity enrichment and mass spectrometry workflow using ac leavable azido-azo-biotin tag [17] conjugated to probetreated MCF7 lysates.B iotinylated proteins were enriched by streptavidin agarose resin and eluted with sodium dithion-ite and resolved with SDS-PAGE( see Figure S8). Protein bands were excised, digested with trypsin, and the released peptides were analyzed by nanoLC-MS/MS.W ei dentified 114 protein targets (!…”

ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures

“…AlkMG has previously been reported to mimic MG both with respect to electrophilic properties as well as it becomes detoxified into the corresponding alkynylated lactate (alkLactate) through the glyoxalase system (Fig. 6d) [33,34]. HEK293 cells where accordingly pretreated with BSO before incubation with 500 μM alkMG for 4 h and then the formation of alkLactate was quantified using UPLC-MS/MS as a measure of GLO1 activity.…”

Non-enzymatic Covalent Modifications as a New Chapter in the Histone Code

“…Finally, the continued development of chemical and biological tools will prompt exciting opportunities for discovery in the field. [80,81] Adduct biotin or fluorescent labeling and subsequent enrichment of modified proteins [78] Adduct fluorescent labeling and subsequent enrichment for proteomics [23] Acylation Adduct biotin or fluorescent labeling and subsequent enrichment of modified proteins for proteomics [25] Capturing and fluorescently visualizing or enriching homocysteine adducts for proteomics or site determination [82] Lipidation Adduct biotin or fluorescent labeling and subsequent enrichment of modified proteins [63,64] Trends in Biochemical Sciences…”

“…For example, two alkynyl-modified MGO probes have been synthesized and used for visualization and enrichment of MGO adducts. The longer of the two was used to characterize the blood MGO proteome, whereas the shorter was used to visualize and enrich histone MGO-glycation NECMs [78,80,81]. Similarly, an azidoribose probe was recently synthesized and applied to track histone ribose glycation.…”