Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Hellbrunnerstraße 34, 5020 Salzburg, Austria.
J Chromatogr A. 2010 Oct 1;1217(40):6159-68. doi: 10.1016/j.chroma.2010.07.044. Epub 2010 Jul 21.
The availability of robust and highly efficient separation methods represents a major requirement for proteome analysis. This study investigated the characteristics of two different gel-free proteomic approaches to the fractionation of proteolytic peptides and intact proteins, respectively, in a first separation dimension. Separation and mass spectrometric detection by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) were performed at the peptide level in both methods. Bottom-up analysis (BU) was carried out employing well established peptide fractionation in the first separation dimension by strong cation-exchange chromatography (SCX), followed by ion-pair reversed-phase chromatography (IP-RPC) in the second dimension. In the semi-top-down approach (STD), which involved intact protein fractionation in the first dimension, the separation mode in both dimensions was IP-RPC utilizing monolithic columns. Application of the two approaches to the proteome analysis of proteins extracted from a tumor tissue revealed that the BU method identified more proteins (1245 in BU versus 920 in STD) while STD analysis offered higher sequence coverage (14.8% in BU versus 17.5% in STD on average). The identification of more basic and larger proteins was slightly favored in the BU approach, most probably due to higher losses of these proteins during intact protein handling and separation in the STD method. A significant degree of complementarity was revealed by an approximately 33% overlap between one BU and STD replicate, while 33% each of the protein identifications were unique to both methods. In the STD method, peptides obtained upon digestion of the proteins contained in fractions of the first separation dimension covered a broad elution window in the second-dimension separation, which demonstrates a high degree of "pseudo-orthogonality" of protein and peptide separation by IP-RPC in both separation dimensions.
强大且高效的分离方法的可用性是蛋白质组分析的主要要求。本研究分别考察了两种不同的凝胶自由蛋白质组学方法在第一分离维度中对蛋白水解肽和完整蛋白质的分离特性。两种方法均在肽水平上通过基质辅助激光解吸/电离串联质谱（MALDI-MS/MS）进行分离和质谱检测。自上而下分析（BU）采用强阳离子交换色谱（SCX）在第一分离维度中进行的成熟肽分级，然后在第二维度中进行离子对反相色谱（IP-RPC）。在涉及完整蛋白质在第一维度中的分级的半自上而下方法（STD）中，两个维度中的分离模式均为利用整体柱的离子对反相色谱。将这两种方法应用于从肿瘤组织中提取的蛋白质的蛋白质组分析表明，BU 方法鉴定出更多的蛋白质（BU 中为 1245 种，STD 中为 920 种），而 STD 分析提供了更高的序列覆盖率（BU 中平均为 14.8%，STD 中为 17.5%）。在 BU 方法中，略微有利于鉴定更多的碱性和较大的蛋白质，这很可能是由于在 STD 方法中完整蛋白质处理和分离过程中这些蛋白质的损失较高。通过一个 BU 和 STD 重复之间约 33%的重叠，揭示了相当大的互补性，而两种方法各自的蛋白质鉴定中各有 33%是独特的。在 STD 方法中，消化第一分离维度的各部分中包含的蛋白质后获得的肽在第二维分离中覆盖了很宽的洗脱窗，这证明了 IP-RPC 在两个分离维度中对蛋白质和肽分离的高度“伪正交性”。
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