Jaffe H, Pant H C
LNC-NINDS Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Nov 17;37(46):16211-24. doi: 10.1021/bi981264p.
A new method for the characterization of serine and threonine phosphorylation sites in proteins has been developed. After modification of a phosphoprotein by beta-elimination/ethanethiol addition and conversion of phosphoserine and phosphothreonine residues to S-ethylcysteinyl or beta-methyl-S-ethylcysteinyl residues, the modified protein was subjected to proteolytic digestion. Resulting digests were analyzed by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry and database searching to identify original phosphorylated residues. The computer program utilized (SEQUEST) is capable of identifying peptides and modified residues from uninterpreted MS/MS spectra, and using this method, all of the five known phosphorylation sites in bovine beta-casein were identified. Application of the method to multiply phosphorylated human high molecular weight neurofilament protein (NF-H) resulted in the identification of 21 peptides and their modified residues and hence, the in vivo phosphorylation sites. These included 26 KSP and 1 KTP site, all of which occur in the KSP repeat C-terminal tail domain (residues 502-823). One site at residue 518 was previously uncharacterized. A novel non-KSP serine at residue 421 near the KLLEGEE region in a IPFSLPE motif was characterized as phosphorylated (or glycosylated). The 27 characterized phosphorylation sites occur at S/TP residues in the following motifs: KSPVKEE, KSPAEAK, KSPEKEE, KSPAEVK, KSPEKAK, KSPPEAK, KSPVKAE, and KTPAKEE. On the basis of kinase consensus sequences, all of these motifs, including the previously unreported KTPAKEE motif, can be phosphorylated by proline-directed kinases. Advantages of the new method vis-a-vis our previously reported method [Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931-3940] include (i) production of diastereomers eluting at different retention times increased the chances of peptide identification, (ii) increased hydrophobicity and hence retention time of the modified peptides, (iii) facilitation of positive ion production, and (iv) increased susceptibility to tryptic digestion as a result of conversion of negatively charged phosphorylated residues to neutral S-ethylcysteine or beta-methyl-S-ethylcysteine residues.
已开发出一种用于鉴定蛋白质中丝氨酸和苏氨酸磷酸化位点的新方法。通过β-消除/添加乙硫醇对磷蛋白进行修饰，并将磷酸丝氨酸和磷酸苏氨酸残基转化为S-乙基半胱氨酰或β-甲基-S-乙基半胱氨酰残基后，对修饰后的蛋白质进行蛋白酶消化。所得消化产物通过微径液相色谱、电喷雾电离串联（MS/MS）离子阱质谱和数据库搜索相结合的方式进行分析，以鉴定原始的磷酸化残基。所使用的计算机程序（SEQUEST）能够从未经解释的MS/MS谱图中鉴定肽段和修饰残基，使用该方法，鉴定出了牛β-酪蛋白中所有五个已知的磷酸化位点。将该方法应用于多重磷酸化的人高分子量神经丝蛋白（NF-H），鉴定出了21个肽段及其修饰残基，从而确定了体内磷酸化位点。这些位点包括26个KSP和1个KTP位点，它们均位于KSP重复C末端尾部结构域（残基502 - 823）。残基518处的一个位点此前未被鉴定。在IPFSLPE基序中靠近KLLEGEE区域的残基421处的一个新的非KSP丝氨酸被鉴定为磷酸化（或糖基化）。所鉴定的27个磷酸化位点出现在以下基序的S/TP残基处：KSPVKEE、KSPAEAK、KSPEKEE、KSPAEVK、KSPEKAK、KSPPEAK、KSPVKAE和KTPAKEE。基于激酶共有序列，所有这些基序，包括此前未报道的KTPAKEE基序，都可被脯氨酸定向激酶磷酸化。与我们之前报道的方法[Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931 - 3940]相比，新方法的优点包括：（i）产生在不同保留时间洗脱的非对映异构体增加了肽段鉴定的机会；（ii）修饰肽段的疏水性增加，从而保留时间延长；（iii）便于产生正离子；（iv）由于带负电荷的磷酸化残基转化为中性的S-乙基半胱氨酸或β-甲基-S-乙基半胱氨酸残基，提高了对胰蛋白酶消化的敏感性。
