Institute for Eye Research, Sydney, Australia.
Exp Eye Res. 2010 Feb;90(2):344-9. doi: 10.1016/j.exer.2009.11.013. Epub 2009 Nov 29.
Proteins are very important components in tears. Their phosphorylation is an important posttranslational modification affecting biological activity. Using proteomic techniques, this study was designed to analyze phosphoproteins found in open eye basal tears from normal human subjects. Proteins in tear samples were separated in 1-dimensional (1D) and 2-dimensional (2D) gels and phosphoproteins were selectively stained with Pro-Q diamond dye before visualization of all proteins using Sypro Ruby. Potential phosphoproteins in 2D gels were identified by liquid chromatography-mass spectrometry (LC-MS/MS) after trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO(2)) columns. The tryptic digests of the tear samples were also analyzed to identify phosphoproteins directly by LC-MS/MS after phosphopeptide enrichment. The major phosphoprotein stained by Pro-Q diamond in the gels and identified by LC-MS/MS from the spots was tear lipocalin. Tear lipocalin was separated into 3 different isoforms and one phosphorylation site (serine at position 24) was identified in one of the isoforms. Prolactin-induced protein, nucleobindin-2 and lipophilin C were also stained with Pro-Q diamond although no phosphorylated peptides from these proteins could be found using LC-MS/MS. Direct analysis of the tear tryptic digests by LC-MS/MS identified a further 12 potential phosphoproteins with tear lipocalin predominant. Four phosphorylation sites (position 24 (serine), 32 (serine), 34 (threonine) and 36 (tyrosine)) were identified for tear lipocalin using this method. These results indicate that tear lipocalin is the predominant phosphoprotein in normal human basal tears. Nucleobindin-2, prolactin-induced protein and lipophilin C also appear to be phosphorylated in basal tear samples.
蛋白质是泪液中非常重要的成分。其磷酸化是影响生物活性的重要翻译后修饰。本研究采用蛋白质组学技术，旨在分析正常人睁眼基础泪液中的磷酸蛋白。泪液样本中的蛋白质在一维（1D）和二维（2D）凝胶中分离，并用 Pro-Q diamond 染料选择性染色磷酸蛋白，然后用 Sypro Ruby 可视化所有蛋白质。通过胰蛋白酶消化和使用二氧化钛（TiO2）柱进行磷酸肽富集后，对 2D 凝胶中的潜在磷酸蛋白进行液相色谱-质谱（LC-MS/MS）鉴定。通过 TiO2 柱进行磷酸肽富集后，直接对 LC-MS/MS 分析泪液样本的胰蛋白酶消化物，以鉴定磷酸蛋白。Pro-Q diamond 染色的凝胶中的主要磷酸蛋白和通过 LC-MS/MS 从斑点中鉴定的主要磷酸蛋白是泪液 lipocalin。泪液 lipocalin 分离成 3 种不同的同工型，其中 1 种同工型鉴定出 1 个磷酸化位点（24 位丝氨酸）。虽然通过 LC-MS/MS 未发现这些蛋白的磷酸肽，但 prolactin-induced protein、nucleobindin-2 和 lipophilin C 也用 Pro-Q diamond 染色。通过 LC-MS/MS 直接分析泪液胰蛋白酶消化物，鉴定出另外 12 种潜在的磷酸蛋白，以泪液 lipocalin 为主。使用该方法鉴定出泪液 lipocalin 的 4 个磷酸化位点（24 位丝氨酸（Ser）、32 位丝氨酸（Ser）、34 位苏氨酸（Thr）和 36 位酪氨酸（Tyr））。这些结果表明，泪液 lipocalin 是正常人基础泪液中的主要磷酸蛋白。核结合蛋白-2、催乳素诱导蛋白和 lipophilin C 似乎也在基础泪液样本中发生磷酸化。
