On studying protein phosphorylation patterns using bottom-up LC-MS/MS:: the case of human α-casein
On studying protein phosphorylation patterns using bottom-up LC-MS/MS:: the case of human α-casein
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DOI：
10.1039/b701902e
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发表时间：
2007-01-01
期刊：
影响因子：
4.2
通讯作者：
Zubarev, Roman A.
中科院分区：
文献类型：
--
作者：
Kjeldsen, Frank;Savitski, Mikhail M.;Zubarev, Roman A.
大多数蛋白质组学研究涉及绘制翻译后修饰图，例如丝氨酸和苏氨酸的磷酸化，目前使用的是“自下而上”的方法。这种方法涉及蛋白质的酶裂解，最常见的是用胰酶，随后的纳米LC-MS/MS。蛋白质中亚磷酸盐的占有率可能会有数量级的不同，因此必须报告每个亚磷酸盐的占有率。为了突出在量化占有率方面的潜在缺陷，来自母乳的α(S1)-酪蛋白被选为代表中等磷酸化蛋白质的模型分子。为此，使用了一名高加索妇女在哺乳第八个月时的母乳。酪蛋白的磷酸化水平被认为对胶束的形成具有重大意义，胶束参与向新生儿运送有价值的钙、磷和其他矿物质。据报道，人类α(S1)-酪蛋白的磷酸化程度比反刍动物酪蛋白低得多，这可能表明酪蛋白在人类中具有不同的功能。因此，揭示人类酪蛋白的磷酸化模式可以揭示其功能。目前的研究发现，与之前的研究相反，人α(S1)-酪蛋白中Ser70和Ser76残基之间的序列区域实际上是磷酸化的。最丰富的磷酸化位点是Ser75，这与已知的乳腺酪蛋白激酶的作用一致。有证据表明该区域存在第二次磷酸化，可能发生在Ser73。早先报道的Ser18和Ser26的磷酸化位置也得到了证实，但没有证实Ser18磷酸化的主导地位。Ser18、Ser26和Ser75的入住率估计分别为(7+/-2)、(20+/-6)和(27+/-9)%。由于磷酸化和非磷酸化多肽之间电离效率的差异，占用率增加了30%的误差。自下而上策略的突出缺陷包括酶对近端酸性和磷酸化残基的敏感性，以及胰蛋白酶多肽的多种异构体的存在，包括意想不到的异构体。演示了前面介绍的PhosTS_Hunter和ModifiComb方法用于避免后一种陷阱的实用性。
Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano-LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, alpha(s1)-casein from human milk was selected as a model molecule representing moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other minerals to the newborn. Human alpha(s1)-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found that the sequence region between the residues Ser70 and Ser76 in human alpha(s1)-casein is in fact phosphorylated, contrary to previous knowledge. The site of the most abundant phosphorylation is Ser75, in agreement with the known action of the mammary gland casein kinase. There is evidence for the second phosphorylation in that region, possibly at Ser73. Earlier reported positions of phosphorylations at Ser18 and Ser26 are also confirmed, but not the dominance of Ser18 phosphorylation. The occupancy rates at Ser18, Ser26 and Ser75 are estimated to be ( 7 +/- 2), ( 20 +/- 6) and ( 27 +/- 9)%, respectively. Owing to differences in the ionization efficiency between phosphorylated and unphosphorylated peptides a 30% error margin is added to the occupancy rates. The highlighted pitfalls of the bottom-up strategy include the sensitivity of enzymes to proximal acidic and phosphorylated residues and the presence of multiple isoforms, including unexpected ones, of the tryptic peptides. The utility of the earlier introduced PhosTS_hunter and ModifiComb approaches for evading the latter pitfall is demonstrated.