Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: An alternative approach to high-resolution peptide separation for shotgun proteome analysis
Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: An alternative approach to high-resolution peptide separation for shotgun proteome analysis
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DOI：
10.1021/pr070424t
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发表时间：
2007-11-01
影响因子：
4.4
通讯作者：
Huber, Christian G.
中科院分区：
文献类型：
--
作者：
Delmotte, Nathanael;Lasaosa, Maria;Huber, Christian G.
建立了一种利用高ph反相高效液相色谱和低ph离子对反相高效液相色谱（RP × IP-RP-HPLC）进行鸟枪蛋白质组分析的二维分离方案，并对其进行了评价。与传统的强阳离子交换x离子对反相（SCX x IP-RP-HPLC）方法相比，RP x IP-RP-HPLC体系的正交度较低，但具有较高的分离效率、更均匀的肽洗脱分布和更易于实验处理等优点。采用电喷雾-电离串联质谱（ESI-MS/MS）技术对多肽片段进行指纹图谱检测和鉴定。该方法可在30%的分析时间内多鉴定约13%的多肽和7%的蛋白质。谷氨酸棒状杆菌的蛋白质组覆盖率为24.9%（745个蛋白质）。结合SCX- x IP-RP-HPLC-ESI-MS/MS和RP- x IP-RP-HPLC-ESI-MS/MS两种方法的鉴定结果，共鉴定出871个蛋白，占谷氨酸酵母基因组注释蛋白总数的29.1%。
A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of. the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.