Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

中文翻译：

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与加州鱼雷乙酰胆碱酯酶四聚体相关的富含脯氨酸的肽。

乙酰胆碱酯酶（AChE）通过水解乙酰胆碱来终止胆碱能神经传递。胶原蛋白尾巴的AChE四聚体是2个基因ACHE和ColQ的产物。AChE四聚体由4个相同的AChE亚基和一个富含多脯氨酸的肽组成，其功能是将4个AChE亚基保持在一起。我们的目标是确定加利福尼亚鱼雷AChE（TcAChE）四聚体中富含多脯氨酸的肽的氨基酸序列，以帮助分析将通过冷冻EM获得的图像。从加州鱼雷电器官中溶解胶原尾AChE，通过胰蛋白酶消化将其转化为300 kDa四聚体，并通过亲和色谱法纯化。通过在沸水浴中使TcAChE四聚体变性，并与二硫苏糖醇还原二硫键，可以释放出富含脯氨酸的多肽。在旋转过滤器上从TcAChE蛋白中分离出氨基甲酰甲基化的肽，然后在高分辨率Orbitrap Fusion Lumos质谱仪上通过液相色谱串联质谱对它们进行分析。在鉴定出的64种胶原尾（ColQ）肽中，有60种来自ColQ N末端附近的富含多脯氨酸的区域。最丰富的富含脯氨酸的肽是SVNKCCLLTPPPPPMFPPPFFTETNILQE，占总质谱信号强度的40％；以及SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK，占总强度的27％。这两种肽的高丰度使其成为胰蛋白酶处理的TcAChE四聚体中富含多脯氨酸的肽主要形式的候选者。