Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
J Proteomics. 2010 Jan 3;73(3):637-48. doi: 10.1016/j.jprot.2009.09.013. Epub 2009 Sep 25.
The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the LMF (i.e., <or=25K) of plasma routinely prepared from normal, healthy volunteers. Four commercially-available filter membranes of similar nominal molecular weight cut-off (NMWC), but differing membrane chemistries and filter orientations (Microcon, Millipore; Centrisart, Sartorius; Amicon Ultra, Millipore; Vivaspin, Sartorius), were evaluated. Of these filtration devices, only the Sartorius Vivaspin tangential membrane, NMWC 20K was effective in the non-retention of M(r)>50K, and recovery and enrichment of low-M(r) components from human plasma. This filter membrane device was further optimized with respect to plasma buffer composition, centrifugal force, duration and temperature. Optimal ultrafiltration conditions were obtained using 100 microL of normal plasma in 10% acetonitrile, and a centrifugation force of 4000x g for 35 min at 20 degrees C. In this LMF, 44 proteins (from 266 unique peptides) were identified using a combination of 1D-SDS-PAGE / nano-LC-MS/MS and a stringent level of identification (FDR <1%). We report the identification of several proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization (HUPO) Plasma Proteome Project datasets. When compared with the low-M(r) human plasma/serum proteome datasets of Zhou et al. (Electrophoresis, 2004. 25, 1289-98), Gundry et al. (Proteomics Clin. Appl., 2007. 1, 73-88) and Villanueva et al. (Anal Chem, 2004. 76, 1560-70), 64% of our identifications (28 proteins) were novel; these include cofilin-1, PPIase A, and the SH3 domain-binding glutamic acid-rich-like protein 3. In addition to intact proteins, many peptide fragments from high-abundance proteins (e.g., fibrinogen, clusterin, Factor XIIIa, transferrin, kinogen-1, and inter-alpha-trypsin inhibitor), presumably derived by ex vivo proteolysis, were observed.
血浆蛋白质组的低分子量馏分（LMF）是生物信息的宝贵来源，尤其是在鉴定疾病的血浆生物标志物方面。在这项研究中，开发了一种基于离心超滤的分离和富集策略，用于常规从正常健康志愿者制备的血浆的 LMF（即，<或=25K）。评估了四种具有相似标称分子量截止值（NMWC）但具有不同膜化学和过滤方向的市售滤膜（Microcon、Millipore；Centrisart、Sartorius；Amicon Ultra、Millipore；Vivaspin、Sartorius）。在这些过滤设备中，只有 Sartorius Vivaspin 切向膜（NMWC 20K）能够有效地保留 M(r)>50K，并且从人血浆中回收和富集低 M(r)成分。进一步优化了该滤膜装置的血浆缓冲液组成、离心力、持续时间和温度。使用 10%乙腈中的 100 μL 正常血浆和 20°C 下 4000x g 离心 35 分钟，获得最佳超滤条件。在这种 LMF 中，使用 1D-SDS-PAGE/nano-LC-MS/MS 和严格的鉴定水平（FDR <1%）鉴定了 44 种蛋白质（来自 266 个独特肽）。我们报告了几种蛋白质（例如，蛋白 KIAA0649（Q9Y4D3）、类风湿因子 D5、丝氨酸蛋白酶抑制剂 A3 和跨膜衔接蛋白 PAG）的鉴定，这些蛋白质以前未在现有的高可信度人类蛋白质组组织（HUPO）血浆蛋白质组项目数据集中报道。与 Zhou 等人的低分子量人血浆/血清蛋白质组数据集（电泳，2004.25，1289-98）、Gundry 等人的（蛋白质组学临床应用，2007.1，73-88）和 Villanueva 等人的（分析化学，2004.76，1560-70）相比，我们的鉴定中有 64%（28 种蛋白质）是新的；其中包括原肌球蛋白-1、PPIase A 和富含谷氨酸的 SH3 结构域结合蛋白 3。除完整蛋白质外，还观察到许多高丰度蛋白质（如纤维蛋白原、簇蛋白、因子 XIIIa、转铁蛋白、激肽原-1 和α-胰蛋白酶抑制剂内肽酶）的肽片段，这些片段可能来自体外蛋白酶解。
