Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry -: Evidence for in vivo doubly phosphorylated dynamin I and dynamin III
Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry -: Evidence for in vivo doubly phosphorylated dynamin I and dynamin III
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DOI：
10.1074/mcp.m300105-mcp200
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发表时间：
2004-05-01
影响因子：
7
通讯作者：
Roepstorff, P
中科院分区：
文献类型：
--
作者：
Larsen, MR;Graham, ME;Roepstorff, P
蛋白质组学中提高质谱法（MS）检测到的肽的数量和质量的常见策略是在分析之前使用反相（RP）色谱法脱盐和浓缩蛋白水解酶。然而，这不允许检测小肽或亲水肽，或亲水性改变的肽，如磷酸肽。我们使用微柱比较RP树脂或石墨粉保留磷酸肽的能力。许多标准的磷酸肽和生物学相关的磷蛋白，发动蛋白I，进行了分析。MS显示，一些磷酸肽不结合RP树脂，但有效地保留在石墨上。那些结合RP树脂的通常从石墨粉末产生强得多的信号。特别是，该方法揭示了双重磷酸化的肽在胰蛋白酶消化的发动蛋白I纯化的大鼠脑神经末梢。该肽的检测大大提高了石墨微量纯化。通过串联MS测序证实了在Ser-774和Ser-778处存在磷酸，而单一磷酸化的肽主要仅在Ser-774上磷酸化。该方法进一步揭示了发动蛋白III中的单磷酸化和双磷酸化肽，类似于发动蛋白I序列。通过串联质谱法在丝氨酸759和丝氨酸763处发现了一对动力蛋白III的磷酸化位点，首次直接确定了动力蛋白I和III的体内磷酸化位点。研究结果表明，MS检测少量磷酸肽的方法有很大的改进，对磷酸蛋白质组学的小型和大型项目都有重大影响。
A common strategy in proteomics to improve the number and quality of peptides detected by mass spectrometry (MS) is to desalt and concentrate proteolytic digests using reversed phase ( RP) chromatography prior to analysis. However, this does not allow for detection of small or hydrophilic peptides, or peptides altered in hydrophilicity such as phosphopeptides. We used microcolumns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analyzed. MS revealed that some phosphopeptides did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve terminals. The detection of this peptide was greatly enhanced by graphite micropurification. Sequencing by tandem MS confirmed the presence of phosphate at both Ser-774 and Ser-778, while a singly phosphorylated peptide was predominantly phosphorylated only on Ser-774. The method further revealed a singly and doubly phosphorylated peptide in dynamin III, analogous to the dynamin I sequence. A pair of dynamin III phosphorylation sites were found at Ser-759 and Ser-763 by tandem MS. The results directly define the in vivo phosphorylation sites in dynamins I and III for the first time. The findings indicate a large improvement in the detection of small amounts of phosphopeptides by MS and the approach has major implications for both small- and large-scale projects in phosphoproteomics.