Characterization of the phosphorylation sites of human high molecular weight neurofilament protein by electrospray ionization tandem mass spectrometry and database searching
Characterization of the phosphorylation sites of human high molecular weight neurofilament protein by electrospray ionization tandem mass spectrometry and database searching
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DOI：
10.1021/bi972518u
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发表时间：
1998-03-17
期刊：
影响因子：
2.9
通讯作者：
Pant, HC
中科院分区：
文献类型：
--
作者：
Jaffe, H;Veeranna;Pant, HC
超磷酸化的高分子神经丝蛋白(NF-H)在其C-末端结构域的赖氨酸-丝氨酸-脯氨酸(KSP)重复序列上表现出广泛的磷酸化。用蛋白水解法、微孔液相色谱、电喷雾电离串联(MS/MS)离子陷阱质谱仪和数据库检索相结合的方法对人核因子-H的特定磷酸化位点进行鉴定。所使用的计算机程序(PEPSEARCH和SEQUEST)能够从未解释的MS/MS谱中识别多肽和磷酸化位点，并利用这些方法鉴定了27个磷酸化多肽及其磷酸化残基。在这些磷酸肽的基础上，鉴定了人核因子-H的38个磷酸化位点。它们包括33个KSP、赖氨酸-苏氨酸-脯氨酸(KTP)或精氨酸-丝氨酸-脯氨酸(RSP)位点和4个非磷酸化位点，它们都存在于KSP重复结构域(残基502-823)，以及在KVPTPEK基序中观察到的一个苏氨酸磷酸化位点。由于未能分离和鉴定相应的磷酸肽，6个KSP位点没有被鉴定，根据酶的特异性，在3个位点观察到丝氨酸和苏氨酸磷酸化的异质性或推测在3个位点发生。根据已鉴定的磷酸化基序(KSPEKEE、KSPVKAE、KSPAEAK、KSPPEAK、KSPEAKT、KSPAEVK和KVPTPEK)，人的NF-H尾部结构域被推测为脯氨酸导向的激酶的底物，苏氨酸磷酸化的KVPTPEK基序表明存在一种新的脯氨酸导向的激酶。
Hyperphosphorylated high molecular weight neurofilament protein (NF-H) exhibits extensive phosphorylation on lysine serine-proline (KSP) repeats in the C-terminal domain of the molecule. Specific phosphorylation sites in human NF-H were identified by proteolytic digestion and analysis of the resulting digests by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry, and database searching. The computer programs utilized (PEPSEARCH and SEQUEST) are capable of identifying peptides and phosphorylation sites from uninterpreted MS/MS spectra, and by use of these methods, 27 phosphopeptides and their phosphorylated residues were identified. On the basis of these phosphopeptides, 38 phosphorylation sites in human NF-H were characterized. These include 33 KSP, lysine-threonine-proline (KTP) or arginine-serine proline (RSP) sites and four unphosphorylated sites, all of which occur in the KSP repeat domain (residues 502-823), and one threonine phosphorylation site observed in a KVPTPEK motif. Six KSP sites were not characterized because of the failure to isolate and identify corresponding phosphopeptides, Heterogeneity in serine and threonine phosphorylation was observed at three sites or deduced to occur at three sites on the basis of enzyme specificity. As a result of the phosphorylated motifs identified (KSPEKEE, KSPVKAE, KSPAEAK, KSPPEAK, KSPEAKT, KSPAEVK, and KVPTPEK), human NF-H tail domain is postulated to be a substrate of proline-directed kinases, The threonine-phosphorylated KVPTPEK motif suggested the existence of a novel proline-directed kinase.