School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
J Proteomics. 2013 Apr 26;82:254-62. doi: 10.1016/j.jprot.2013.02.008. Epub 2013 Feb 26.
ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compared the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionates peptides according to their pI and GRAVY values. These properties remains but becomes less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY values of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP is good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives.
For in-depth proteomic analysis of a cell, tissue and plasma, multidimensional liquid chromatography (MDLC) is still necessary to reduce sample complexity for improving analytical dynamic range and proteome coverage. This work conducts a direct comparison of three promising first-dimensional proteome fractionation methods. They are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC. ERLIC is good at analyzing basic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI<4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods.
已有报道称，ERLIC 和高 pH 值反向相（Hp-RP）在蛋白质组分离方面是强阳离子交换（SCX）的很有前途的替代方法。在这里，我们比较了 ERLIC、串联 ERLIC 和串联 Hp-RP 在蛋白质组分析中的性能。这三种策略的蛋白质鉴定结果相当，但通过两种串联方法鉴定出的独特肽更多，从而使平均蛋白质序列覆盖率显著增加。与基于色谱图的相邻馏分的合并相比，在间隔时间内合并馏分可使肽在每个馏分中的分布更加均匀。ERLIC 根据其 pI 和 GRAVY 值对肽进行分级。这些特性仍然存在，但在串联 ERLIC 中变得不那么明显。相比之下，在串联 Hp-RP 中，每个馏分的肽的平均 pI 和 GRAVY 值相当。在这三种策略中，ERLIC 最擅长鉴定 pI>9 的肽，而串联 Hp-RP 则擅长鉴定 pI<4 的肽。当基本或酸性肽/蛋白质是分析目标时，这些优势是有用的。ERLIC 鉴定碱性肽的能力似乎是由于它们与酸性肽的有效分离。这项研究有助于根据具体目标选择适当的分级策略。
对于细胞、组织和血浆的深入蛋白质组分析，多维液相色谱（MDLC）仍然是必要的，以减少样品复杂性，提高分析动态范围和蛋白质组覆盖率。这项工作对三种很有前途的一维蛋白质组分级方法进行了直接比较。它们在鉴定蛋白质方面相当，但串联 ERLIC 和串联 Hp-RP 鉴定的独特肽明显多于 ERLIC。ERLIC 擅长分析碱性肽，而串联 Hp-RP 在分析 pI<4 的酸性肽方面表现最好。这将有助于根据特定需求选择适当的肽分级策略。由于不同方法之间存在互补效应，因此可以使用不同的分级策略组合来增加序列覆盖率和蛋白质鉴定数量。
