Global phosphoproteome of HT-29 human colon adenocarcinoma cells
Global phosphoproteome of HT-29 human colon adenocarcinoma cells
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DOI：
10.1021/pr050048h
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发表时间：
2005-07-01
影响因子：
4.4
通讯作者：
White, FM
中科院分区：
文献类型：
--
作者：
Kim, JE;Tannenbaum, SR;White, FM
多种细胞刺激触发的细胞信号级联中的磷酸化事件调节蛋白质功能，导致多种细胞结果，包括细胞分裂、生长、死亡和分化。由于信号蛋白的突变或过度表达导致蛋白磷酸化的异常调节往往导致各种疾病状态。本文利用固定化金属亲和层析（IMAC）结合液相色谱-串联质谱（MS/MS）技术，从人结肠癌细胞系HT-29中鉴定出蛋白磷酸化位点。在本研究中，从HT-29全细胞裂解物中提取的蛋白质用胰蛋白酶消化，所得肽上的羧酸基转化为甲酯。利用Fe3+螯合金属亲和树脂富集衍生化的磷酸化肽。采用高效液相色谱（HPLC）分离IMAC保留的磷酸肽，并采用电喷雾电离-四极杆飞行时间（ESI-Q-TOF）质谱分析。从116个蛋白中鉴定出238个磷酸化位点，其中213个可以明确定位于单个残基。通过手工检查MS/MS光谱确认肽鉴定和磷酸化位点分配。在我们的研究结果中发现的许多磷酸化位点以前在科学文献中没有描述过。我们试图通过使用Scansite （http://scansite.mit.edu）搜索潜在的激酶基序，并从Pattern Explorer （http://scansite.mit.edu/pe）获得有关激酶底物选择性的信息，将功能归因于在这项工作中确定的位点。本实验中确定的蛋白磷酸化位点列表提供了正常（异步）细胞培养条件下磷酸化蛋白的广泛信息。本研究中确定的位点可以作为替代生物标记物来评估来自不同细胞状态和外源刺激的选定激酶和信号通路的活性。
Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated pepticles were enriched using Fe3+-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.