Ahmed Saleh, Altman Jeremy, Jones Garrett, Lee Tae Jin, Robertson Danielle M, Zhi Wenbo, Sharma Shruti, Sharma Ashok
Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta University, Augusta, GA, 30912, USA.
Department of Ophthalmology, UT Southwestern Medical Center, Dallas, TX, 75390, USA.
Exp Eye Res. 2025 Feb;251:110231. doi: 10.1016/j.exer.2025.110231. Epub 2025 Jan 4.
Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.
Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.
We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R = 0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants.
This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.
角蛋白污染是质谱蛋白质组学分析中的常见问题，尤其是在自下而上的质谱分析中。本研究的目的是确定泪液蛋白质组分析过程中引入的蛋白质污染物。
使用泪液试纸条收集人泪液样本。对空白泪液试纸条和泪液样本进行液相色谱-串联质谱（LC-MS/MS）蛋白质组分析，空瓶用作评估环境污染物蛋白质的对照。
我们检测到26种污染物蛋白质（18种角蛋白和8种非角蛋白）。总蛋白质污染的98.2%可归因于9种角蛋白，包括角蛋白10（KRT-10，23.6%）、角蛋白1（KRT-1，23.5%）、角蛋白2（KRT-2，15.7%）、角蛋白14（KRT-14，7.6%）、角蛋白16（KRT-16，7.0%）、角蛋白5（KRT-5，6.1%）、角蛋白9（KRT-9，5.9%）、角蛋白6B（KRT-6B，4.6%）和角蛋白6A（KRT-6A，4.3%）。与空白泪液试纸条和对照（空瓶）的蛋白质组图谱比较发现有很强的相关性（R = 0.9753），表明这些蛋白质不是来自空白泪液试纸条，而是环境污染物。另一方面，包括角蛋白19、角蛋白13、角蛋白4、角蛋白7、角蛋白15、角蛋白8和角蛋白18在内的几种角蛋白存在于泪液中，但在空白试纸条中未检测到或可忽略不计。另一组角蛋白，包括角蛋白5、角蛋白6A、角蛋白14、角蛋白16和角蛋白17，被鉴定为泪液成分以及环境污染物。
本研究揭示了质谱分析中9种主要的污染角蛋白。其他几种角蛋白被鉴定为泪液成分。使用质谱法准确分析泪液需要进行背景扣除。
