**1. Introduction**

In developed countries, cataracts are one of the most common causes of blindness [1]. They are classified by cause as age-related cataracts, pediatric cataracts, and cataracts secondary to other causes. As shown by many studies, age is the biggest risk factor [2,3]. Considering the location of opacification within the lens, cataracts are divided into three major types: nuclear, cortical, and posterior subcapsular cataracts. Cataract development can be caused by many other risk factors, including environmental factors and genetic changes [4]. Diabetes mellitus (DM), long-term use of corticosteroids, cigarette smoking,

**Citation:** Chang, W.-C.; Lee, C.-H.; Chiou, S.-H.; Liao, C.-C.; Cheng, C.-W. Proteomic Analysis of Aqueous Humor Proteins in Association with Cataract Risks: Diabetes and Smoking. *J. Clin. Med.* **2021**, *10*, 5731. https://doi.org/10.3390/jcm10245731

Academic Editor: Nobuyuki Shoji

Received: 9 November 2021 Accepted: 5 December 2021 Published: 7 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

prolonged exposure to ultraviolet light, and alcohol abuse are well-known risk factors [2]. Cigarette smoking is a risk factor for nuclear and posterior subcapsular cataracts [4]. DM was identified as a common cause of posterior subcapsular and cortical cataracts [5,6]. Increased age is a risk factor for the development of all types of cataracts. Throughout life, a high myopia of over −6.0 D is associated with nuclear cataracts and posterior subcapsular cataracts [7]. Other causes of cataracts include mechanical trauma, chemical injury, electrical injury, radiation, and certain medications. However, the underlying cataractogenic mechanisms of cataract development are still not well documented, with many still being investigated. Proteomics analysis is an extensively used technique to discover changes in protein levels in tissues and cells. Recent proteomic studies in cataract disease of the human aqueous humor (AH) revealed multiple proteins of interest in patients [8–12]. Ji et al. [13] used isobaric tags for the relative and absolute quantitation (iTRAQ) methodology to compare AH protein profiles among high myopia, glaucoma, and vitrectomy surgery patients, and controls. They identified multiple candidate protein biomarkers associated with cataract development in each group. Furthermore, Kim et al. [14] analyzed the aqueous proteome from age-related macular degeneration (AMD) patients and non-AMD cataract controls to identify novel pathogenic proteins that are useful as potential clinical biomarkers. The differential expressions of three proteins were reported in the AH of AMD patients compared with those of cataract controls. Those studies used a good model that inspired a new idea for us of using proteomics to discuss different risk factors of cataract formation. To our knowledge, there has been no previous investigation of different cataract risk factors by comparing proteomic evidence. We used proteomics to discover the pathogenesis of different cataract risks and to possibly identify candidate biomarker proteins identified in patients predisposed to this condition. In this study, we employed Nanoflow ultrahigh-performance liquid chromatography–tandem mass spectrometry (n-UPLC-MS/MS) to examine the protein compositions of aqueous solutions obtained from human cataract eyes of patients who had a single risk factor of either DM or cigarette smoking, those who had double risk factors of DM and cigarette smoking, and aged-matched cataract controls (with neither risk factor). This sensitive proteomics approach could help examine the underlying pathophysiology of cataract formation using relatively scarce amounts of aqueous samples, thereby favoring the methodological approach for this investigation. This study may reveal valuable insights into the molecular changes in the AH in the course of cataract pathogenesis.

#### **2. Materials and Methods**
