Ouidir Tassadit, Jarnier Frédérique, Cosette Pascal, Jouenne Thierry, Hardouin Julie
CNRS, UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, F-76820 Mont-Saint-Aignan, France; Normandy Univ, UR, France; PISSARO Proteomic Facility, IRIB, F-76820 Mont-Saint-Aignan, France.
Normandy Univ, UR, France; PISSARO Proteomic Facility, IRIB, F-76820 Mont-Saint-Aignan, France.
J Proteomics. 2015 Jan 30;114:214-25. doi: 10.1016/j.jprot.2014.11.006. Epub 2014 Nov 21.
Even though protein initiator methionine excision (NME) and N-terminal acetylation (NTA) have been relatively well investigated in eukaryotic proteomes, few studies were dedicated to these modifications in bacteria up to now. In this work, we investigated, for the first time, the N-terminal proteome of the bacterium Pseudomonas aeruginosa PA14 by studying the NME and NTA processes using proteomic approaches. For NME, most of proteins had their initiator Met cleaved (63%) and the nature of the penultimate residue seems to be essential for this cleavage. Concerning NTA, two methods were applied (protein fractionation and peptide enrichment). This allowed us to identify 117 Nα-acetylated proteins, among them 113 have not yet been described as modified in bacteria. Most often, the non-acetylated form was over-represented compared to the acetylated form, arguing that this latter was a minor part of the total abundance of a given protein. Furthermore, some proteins with acetylated initiator methionine were observed. The present work significantly enlarges the number of N-terminally modified proteins in bacteria and confirms that these modifications are a general and fundamental process, not only restricted to eukaryotes.
Protein modifications in prokaryotes have been detected more recently than in eukaryotes. Methionine cleavage and N-terminal acetylation are two common protein N-terminal modifications. Despite their importance in bacterial processes, they are less investigated. The characterization of N-terminal acetylation in bacteria is a challenge because no antibody exists and it is a less frequent modification than in eukaryotes. We used proteomic approaches (enrichment, fractionation, nanoLC-MS/MS, and bioinformatic analyses) to investigate the N-terminal methionine excision and to profile the N-terminal acetylome of P. aeruginosa strain PA14. From our results, around 60% of the proteins had their iMet cleaved. In total, 117 proteins were identified constituting the largest dataset in prokaryotes. Among them, proteins kept their initiator methionine and were acetylated. These results may facilitate the design of experiments to better understand the role of acetylation at the protein N-terminus of P. aeruginosa PA14.
尽管蛋白质起始甲硫氨酸切除（NME）和N端乙酰化（NTA）在真核蛋白质组中已得到相对充分的研究，但迄今为止，针对细菌中这些修饰的研究却很少。在这项工作中，我们首次通过蛋白质组学方法研究铜绿假单胞菌PA14的NME和NTA过程，从而对其N端蛋白质组进行了研究。对于NME，大多数蛋白质的起始甲硫氨酸被切割（63%），倒数第二个残基的性质似乎对这种切割至关重要。关于NTA，我们应用了两种方法（蛋白质分级分离和肽富集）。这使我们能够鉴定出117种Nα-乙酰化蛋白质，其中113种在细菌中尚未被描述为有修饰。通常，与乙酰化形式相比，非乙酰化形式的含量过高，这表明后者只是给定蛋白质总丰度的一小部分。此外，还观察到一些起始甲硫氨酸被乙酰化的蛋白质。本研究显著增加了细菌中N端修饰蛋白质的数量，并证实这些修饰是一个普遍且基本的过程，并不局限于真核生物。
原核生物中的蛋白质修饰比真核生物中发现得更晚。甲硫氨酸切割和N端乙酰化是两种常见的蛋白质N端修饰。尽管它们在细菌过程中很重要，但对其研究较少。细菌中N端乙酰化的表征是一项挑战，因为不存在相应抗体，而且这种修饰比在真核生物中更不常见。我们使用蛋白质组学方法（富集、分级分离、纳升液相色谱-串联质谱以及生物信息学分析）来研究N端甲硫氨酸切除，并描绘铜绿假单胞菌PA14菌株的N端乙酰化蛋白质组。根据我们的结果，约60%的蛋白质的起始甲硫氨酸被切割。总共鉴定出117种蛋白质，构成了原核生物中最大的数据集。其中，一些蛋白质保留了起始甲硫氨酸并被乙酰化。这些结果可能有助于设计实验，以更好地理解铜绿假单胞菌PA14蛋白质N端乙酰化的作用。
