Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae
Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae
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DOI：
10.1038/nbt0302-301
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发表时间：
2002-03-01
影响因子：
46.9
通讯作者：
White, FM
中科院分区：
文献类型：
--
作者：
Ficarro, SB;McCleland, ML;White, FM
蛋白激酶由2000多个基因编码，因此构成了人类基因组中最大的单一酶家族。事实上，大多数细胞过程是由丝氨酸、苏氨酸和酪氨酸残基上蛋白质的可逆磷酸化调控的(1)。至少30%的蛋白质被认为含有共价结合的磷酸盐。尽管这种修饰的重要性和广泛发生，蛋白质磷酸化位点的鉴定仍然是一个挑战，即使是在高度纯化的蛋白质上进行(2)。本文报道了一种方法，该方法可以在单个实验中从全细胞裂解物中表征大多数（如果不是全部）磷酸化蛋白。蛋白质用胰蛋白酶消化，得到的肽转化为甲酯，通过固定化金属亲和层析（IMAC）富集磷酸肽（3,4），并通过纳米流高效液相色谱/电喷雾电离质谱分析。当将该方法应用于分析酿酒酵母的全细胞裂解物时，检测到超过1000个磷酸肽。共确定了216个肽序列，确定了383个磷酸化位点。其中60个是单磷酸化，145个是双磷酸化，11个是三磷酸化。与文献比较发现，这些位点中有18个是先前确定的，包括来自两个有丝分裂原活化蛋白（MAP）激酶激活环的双磷酸化基序pTXpY。我们注意到，该方法可以很容易地扩展到显示和量化两种不同细胞系统中磷酸化蛋白的差异表达，因此证明了一种将“磷酸化谱”作为细胞状态测量的方法。
Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues(1). At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein(2). Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC)(3,4), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.