Hsp27 is ubiquitously expressed throughout the human body. We have shown that Hsp27 is particularly vulnerable to MGO modification in kidney mesangial cells. Others have shown a similar vulnerability of Hsp27 in other cell types. Furthermore, we showed that the chaperone and anti-apoptotic functions of Hsp27 were improved after its modification by MGO. Thus, Hsp27 appears to be a prime target for MGO modification, and consequently, its function could be altered in cells. Altogether, it is clear now that MGO modification of the small heat shock proteins results in an improvement in their key functions. Whether the improvement in the chaperone function of small heat shock proteins occurs via modification of a conserved arginine residue and whether physiological levels of MGO could improve the chaperone function through a hydroimidazolone modification is not known. In this study, we modified human Hsp27 and aA- and aB-crystallin with 2–10 mM MGO and identified hydroimidazolone AGEs using mass spectrometry. Interestingly, the only conserved arginine residue that was modified to hydroimidazolone by MGO was R12 in all three proteins. To determine if the hydroimidazolone modification of this arginine residue is responsible for the improvement of the chaperone function, we replaced R12 with alanine and explored the effect of this mutation on the structure and chaperone function of Hsp27 and aA- and aB-crystallin. MGO is derived mostly from triose phosphate intermediates of glycolysis by non-enzymatic mechanisms in vivo. It is a major precursor of AGEs in tissue proteins. In previous studies, we have shown that MGO modifications of small heat shock proteins, such as aA-crystallin and Hsp27, enhanced their chaperone function. In this study, our primary goal was to determine whether a similar increase in the chaperone function occurred with physiological levels of MGO and to determine whether a modification of the conserved R12 to hydroimidazolone contributed to the increased chaperone function. We first LEE011 determined the “first hit” arginine residues for modification to hydroimidazolone. To accomplish this, we modified the proteins with 2, 5 and 10 mM of MGO. With 2 mM MGO, we found that 6, 6 and 8 arginine residues were modified to hydroimidazolone in aA- and aB-crystallin and Hsp27, respectively. With 10 mM of MGO, this modification reached 10, 8 and 10 arginine residues in the three respective proteins. R12 was the only common residues among the three proteins converted to hydroimidazolone with 2 mM MGO, which suggested that in small heat shock proteins, R12 is the most susceptible for modification to hydroimidazolone by MGO. Notably, a previous study detected a modification of R12 in human lens aA-crystallin that had a molecular weight identical to hydroimidazolone. The modification of arginine residues to hydroimidazolone converts the positive charge on arginine to a neutral charge. Previously, we reported that the substitution of MGO-modifiable arginine residues with neutral alanine residues enhanced the chaperone function of aA-crystallin, similarly to MGO-modification. Because R12 is the most susceptible arginine for MGO modification, we sought to determine if the chaperone function would be improved if it was replaced with alanine. Our results also demonstrated that TNS binding sites are different than the client protein binding sites in all three proteins.