Increasing proteome coverage for gel‐based human tear proteome maps: towards a more comprehensive profiling

Abstract

In situations where the molecular mechanism of many ocular disorders is unknown, owing to the difficulties associated with sampling from ocular tissues, human tear film can be a promising medium in ophthalmic research. The present study demonstrates an in-depth gel-based proteome optimization survey to approach more appropriate and efficient systems in various situations such as normal and dry-eye syndromes. Therefore, systematic and statistical evaluations were performed on different preparation methods, including acetone, acetone-methanol, chloroform-methanol-water, trichloroacetic acid (TCA)-acetone, tri-n-butylphosphate-acetone-methanol precipitations and ammonium sulfate fractionation at three different percentages of saturations (50, 70 and 90%). Methods were compared quantitatively on both one- and two-dimensional patterns. Some important parameters such as total protein recovery yield, densitometric analysis of some protein contaminants, banding patterns and total spot numbers along with statistical models for proper clustering were considered. Findings revealed noticeable impacts of preparation methods on all aspects of gel-based separations as well as recovery yield (ranging from 5.29 ± 0.96 to 22.56 ± 1.77 µg/mm) and banding and pattern resolution. In addition to all these, the most important point is that the total protein spot number on the final two-dimensional patterns (varied from 528.00 ± 19.00 to 657.00 ± 21.52 for different methods) were also noticeably increased in comparison with previously published reports (maximum of 250 spots), which is essential for a more comprehensive analysis. Increasing the proteome coverage in the present study is supposed to originate from improved solubility and effective rehydration during the sample application and isoelectric focusing (IEF) procedure along with proper sample preparation.

Dynamic interfacial properties of human tear-lipid films and their interactions with model-tear proteins in vitro

“…Tear aqueous contains 0.9-1.0 wt.% of inorganic salts, akin to lymph and blood plasma, and up to 7 mg/ml of the numerous proteins, including a vast variety of enzymes, immunoglobulins, glycoproteins, etc. The total number of protein-species identified in human tears has reached 1543 and is still growing [13][14][15]. Lysozyme (2-4 mg/ml), lactoferrin (1.2-3 mg/ml) and lipocalin (1-2 mg/ml) are found among the most abundant proteins [14].…”

“…The total number of protein-species identified in human tears has reached 1543 and is still growing [13][14][15]. Lysozyme (2-4 mg/ml), lactoferrin (1.2-3 mg/ml) and lipocalin (1-2 mg/ml) are found among the most abundant proteins [14].…”

“…The human tear film is a very complex biological system and its composition must be delicately balanced in order to maintain its optimal functions [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. The interactions between tear-lipid films and tearaqueous constituents (e.g., natural tear proteins, or ingredients added in topical ophthalmic medications or lens-care solutions) are important and likely to influence overall tear-film stability [25,26,62].…”

Tear film proteome in age-related macular degeneration

“…It consists of three layers: the outermost lipid layer, the intermediate aqueous layer, and the innermost mucus layer. The tear film is produced by lacrimal glands and Meibomian glands, accessory glands, and goblet cells [ 19 ]. Since it is readily accessible, it has been a target in proteomic programs investigating various ocular and systemic diseases such as dry eye syndrome [ 20 – 22 ], diabetes [ 23 , 24 ], Parkinson’s disease, multiple sclerosis, or cancer [ 25 ].…”

Genetics of congenital cataract, its diagnosis and therapeutics

“…If the physicians notice the cataract, then they recommend clinical involvement. For optical development, the time of surgery is critical [43]. Abnormalities such as ocular hypoplasia and early or late clinical difficulties for example inflammation and glaucoma have an adverse effect on vision in children experiencing congenital cataract surgery.…”