Kim Ji-Eun, Tannenbaum Steven R, White Forest M
Biological Engineering Division, Massachusetts Institute of Technology, 77 Massassachusetts Avenue, Cambridge, MA 02139, USA.
J Proteome Res. 2005 Jul-Aug;4(4):1339-46. doi: 10.1021/pr050048h.
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 peptides were enriched using Fe(3+)-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.
由多种细胞刺激引发的细胞信号级联中的磷酸化事件调节蛋白质功能，导致包括细胞分裂、生长、死亡和分化在内的多种细胞结果。由于信号蛋白的突变或过表达导致的蛋白质磷酸化异常调节通常会导致各种疾病状态。我们在此提供一份通过固定金属亲和色谱（IMAC）结合液相色谱（LC）-串联质谱（MS/MS）分析从HT-29人结肠腺癌细胞系中鉴定出的蛋白质磷酸化位点列表。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，所得肽段上的羧基被转化为甲酯。使用Fe(3+)螯合金属亲和树脂富集衍生化的磷酸化肽段。通过IMAC保留的磷酸肽通过高效液相色谱（HPLC）分离，并用电喷雾电离-四极杆-飞行时间（ESI-Q-TOF）质谱分析。我们通过使用MASCOT在人蛋白质数据库中搜索MS/MS谱图，从116种蛋白质中鉴定出238个磷酸化位点，其中213个可以明确地定位到单个残基。通过人工检查MS/MS谱图确认肽段鉴定和磷酸化位点分配。我们结果中鉴定出的许多磷酸化位点在以前的科学文献中尚未描述。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）获得激酶底物选择性信息，来赋予本研究中鉴定出的位点功能。本实验中鉴定出的蛋白质磷酸化位点列表提供了关于正常（异步）细胞培养条件下磷酸化蛋白质的广泛信息。本研究中鉴定出的位点可作为替代生物标志物，以评估来自不同细胞状态和外源性刺激的选定激酶和信号通路的活性。
