University of Southern Denmark, Campusvej 55, Odense M, Denmark.
Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Mol Cell Proteomics. 2020 Jan;19(1):167-180. doi: 10.1074/mcp.RA119.001700. Epub 2019 Nov 21.
is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.
是慢性牙周炎的关键病原体，最近通过肽基精氨酸脱亚氨酶的活性在牙周组织中产生瓜氨酸化表位，在类风湿关节炎的发病机制中被机械地联系起来。在这个项目中，使用基于 HFBA 的二维分离系统结合 LC-MS 分析了来自 W83 野生型（WT）、肽基精氨酸脱亚氨酶缺失突变体（ΔPPAD）和表达具有活性位点半胱氨酸突变为丙氨酸的 PPAD 的突变株的外膜囊泡（OMV）。为了最佳和阳性鉴定和验证瓜氨酸化肽和蛋白质，使用了高分辨率质谱仪和严格的 MS 搜索标准。这可能会影响鉴定的瓜氨酸化总数，但增加了验证的可信度。新的二维分离系统被证明可以增强验证的强度，并且结合使用内部构建程序，我们建立了一种快速简便的半自动化（手动）瓜氨酸化肽验证方法。对于 WT OMV，我们鉴定了 78 种瓜氨酸化蛋白，总共有 161 个瓜氨酸化位点。值得注意的是，与 OMV 形成的机制一致，大多数（78 个中的 51 个）瓜氨酸化蛋白被预测通过内膜输出，并存在于周质或被转运到细菌表面。瓜氨酸化表面蛋白可能有助于类风湿关节炎的发病机制。对于 C351A-OMV，只发现了一个瓜氨酸化位点，而对于 ΔPPAD-OMV 则没有鉴定到瓜氨酸化，从而验证了我们的瓜氨酸化肽鉴定方法的无偏倚性质。
