Gundry Rebekah L, Fu Qin, Jelinek Christine A, Van Eyk Jennifer E, Cotter Robert J
Department of Pharmacology and Molecular Sciences, Middle Atlantic Mass Spectrometry Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Proteomics Clin Appl. 2007 Jan 1;1(1):73-88. doi: 10.1002/prca.200600276.
The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti-albumin antibody (anti-HSA) affinity chromatography and SEC were used to isolate albumin-bound proteins. RP-HPLC and 1-D SDS-PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.
去除白蛋白和其他高丰度蛋白质是血清和血浆蛋白质组分析中的常规第一步。然而，由于白蛋白可以结合蛋白质和肽，因此人们普遍关注去除白蛋白后血清蛋白质组会如何变化。为了解决这一问题，本研究旨在鉴定在去除白蛋白过程中从血清中去除的蛋白质和肽；确定其中哪些与白蛋白结合（而非共纯化），以及结合的蛋白质是完整蛋白质还是肽片段。采用包括抗白蛋白抗体（抗HSA）亲和色谱和尺寸排阻色谱（SEC）在内的顺序独立分析方法来分离与白蛋白结合的蛋白质。然后使用反相高效液相色谱（RP-HPLC）和一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳（1-D SDS-PAGE）在通过串联质谱（MS/MS）鉴定之前进一步分离蛋白质。最后，将全蛋白分子量（MW）质谱测量结果与通过质谱获得的蛋白质覆盖率相结合，以评估结合的蛋白质是完整的还是肽片段。综合多种方法的结果，发现有35种蛋白质与白蛋白相关，其中24种是完整的，它们包括已知的高丰度和低丰度蛋白质。
