Tirumalai Radhakrishna S, Chan King C, Prieto DaRue A, Issaq Haleem J, Conrads Thomas P, Veenstra Timothy D
SAIC-Frederick Inc., Laboratory of Proteomics and Analytical Technologies, Mass Spectrometry Center, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USA.
Mol Cell Proteomics. 2003 Oct;2(10):1096-103. doi: 10.1074/mcp.M300031-MCP200. Epub 2003 Aug 13.
Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.
血清可能携带着重要的组织学信息档案，其测定有助于改善疾病的早期检测。然而，由于血清中蛋白质/肽成分的动态浓度范围很宽，血清分析在分析上具有挑战性，因此在质谱分析之前需要进行广泛的分级分离。低分子量（LMW）血清蛋白质组是指已去除白蛋白、免疫球蛋白、转铁蛋白和脂蛋白等高分子量蛋白质后的蛋白质/肽部分。这个低分子量部分由几类生理上重要的蛋白质组成，如细胞因子、趋化因子、肽激素以及较大蛋白质的蛋白水解片段。通过血清的离心超滤去除大量的组成蛋白，从而富集低分子量蛋白质/肽。由于已知白蛋白在循环系统中结合并运输小分子和肽，所以离心超滤是在影响蛋白质-蛋白质相互作用破坏的溶剂条件下进行的。低分子量血清蛋白质组样品用胰蛋白酶消化，通过强阳离子交换色谱分级分离，并通过微毛细管反相液相色谱与电喷雾电离串联质谱在线联用进行分析。串联质谱分析鉴定出了340多种人血清蛋白；然而，未观察到来自血清白蛋白的单个肽段。鉴定出的大量蛋白质证明了该方法去除大量丰富蛋白质和富集低分子量血清蛋白质组的有效性。
