Davidsson P, Folkesson S, Christiansson M, Lindbjer M, Dellheden B, Blennow K, Westman-Brinkmalm A
Department of Clinical Neuroscience, Experimental Neuroscience Section, Göteborg University, Sahlgrenska University Hospital/Mölndal, Sweden.
Rapid Commun Mass Spectrom. 2002;16(22):2083-8. doi: 10.1002/rcm.834.
Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.
脑脊液（CSF）与大脑紧密相邻，脑脊液蛋白质组成的变化可能表明神经退行性疾病中大脑蛋白质表达的改变。由于大多数脑脊液蛋白质来源于血浆，往往会掩盖丰度较低的蛋白质，因此分析脑脊液中脑特异性蛋白质变得复杂。通过在二维凝胶电泳（2-DE）之前采用预分级步骤，可以富集丰度较低的蛋白质，并能在诸如脑脊液等复杂蛋白质组中检测到它们。我们开发了一种方法，其中使用液相等电聚焦（IEF）对单个脑脊液样本进行预分级；然后在SYPRO-Ruby染色的二维凝胶上分析选定的IEF级分，最终通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）进行蛋白质鉴定。为了优化二维凝胶上蛋白质斑点的聚焦，将液相IEF中的两性电解质浓度降至最低，并增加第一维的聚焦时间。当比较单个预分级和未分级脑脊液样本的二维凝胶时，很明显，脑脊液预分级后单个蛋白质斑点更大且含有更多蛋白质。一般来说，与脑脊液的直接二维电泳分离相比，预分级脑脊液的二维凝胶中也检测到更多的蛋白质斑点。在预分级的脑脊液中鉴定出了几种蛋白质，包括胱抑素C、IgM-κ、血红素结合蛋白、乙酰辅酶A羧化酶-α和α-1-酸性糖蛋白，但在未分级的脑脊液中未鉴定出。翻译后修饰蛋白质的低丰度形式，例如α-1-酸性糖蛋白和α-2-HS糖蛋白，可以被富集，从而在二维凝胶上得到更好的分离和检测。液相IEF作为二维电泳之前的预分级步骤，降低了样品复杂性，便于检测丰度较低的蛋白质组分，增加了蛋白质上样量以及每个凝胶斑点中用于MALDI-MS分析的蛋白质量。
