High efficient large scale proteome analysis strategies with serially coupled long microcolumn

Comprehensive analysis of whole proteomes is an extraordinary challenge. LC/MS/MS is commonly used to separate peptides digested from proteomes extracted from cells or tissues. To increase protein identification capacity, improvement on separation efficiency and peak capacity of HPLC and sample fractionation to reduce sample complexity are two effective ways. Increasing column length is one of the effective solutions to improve the separation capacity of &#61549;RPLC. However, it was hard to prepare a long microcolumn due to high backpressure generated during packing procedure. In our recent work, through connecting microcolumns of 5, 10 and 15 cm length via unions with minimal dead volume, long microcolumns with lengths up to 30 cm were obtained, and applied for analyzing protein digests. With a 30 cm long microcolumn (300 &#61549;m i.d.), 318 proteins extracted from E. Coli were identified by &#61549;RPLC-ESI MS/MS (LCQ), compared with 70 proteins by a 5 cm long microcolumn. In addition, such a column was applied in 2-D SCX-RPLC/MS/MS for peptides separation and protein identification. Compared to that obtained with normal 10 cm-long &#61549;RPLC column, higher peak capacity (2365 vs 2015) and more identified proteins or protein groups (1836 vs 1358) with FDR<1% were obtained within shorter time (26.7 h vs 39 h). Furthermore, protein pre-fractionation by preparative microscale solution isoelectric focusing (PMSIEF), peptide separation by &#61549;RPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS, were combined, and applied for high resolution separation and high sensitive detection of proteins extracted from E. coli. By comparison with that identified only by &#61549;RPLC-MS/MS with serially coupled long microcolumn, the identified protein number was increased by 3.53 times (835/236), and the Mws and pIs ranges were extended (2563.0 Da to 219155.7 Da vs 2563.0 Da to 163837.0, 3.98 to 11.81 vs 4.01 to 11.42). In addition, a larger scale proteome analysis by combining PMSIEF fractionation technology with 2-D SCX-RPLC/MS/MS with a serially coupled long &#61549;RPLC column was performed, and more proteins were identified

Integrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identification

Integrated microfluidic system for proteomic analysis consisting of on-line protein digestion, peptides separation and identificatio

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱（Ti 4＋-IMAC）整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/LNaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti 4＋-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选

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针对化学蛋白质组学在筛选非水溶性药物的靶蛋白中存在的问题,建立了以非水溶性药物颗粒为载体的靶蛋白筛选方法.通过避免药物固定化,不仅可以保留全部的药物官能团,而且减少了蛋白质在固载基质上的非特异性吸附,可提高获得数据的可信度.将非溶性药物地塞米松颗粒直接与人小细胞肺癌H446细胞提取蛋白质通过间歇性振荡孵育24h,然后采用缓冲液清洗药物颗粒,最后对药物颗粒特异性结合的蛋白质进行酶解和分离鉴定.结果表明,筛选出41个潜在的药物靶蛋白,参与了与DEX药物作用机理相关的多种蛋白质代谢通路和糖代谢通路,同时还发现部分蛋白质参与了帕金森症疾病过程