重新审视蛋白质反相色谱法在自下而上蛋白质组学中的应用

IF 3.8 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Proteome Research Pub Date : 2024-09-18 DOI:10.1021/acs.jproteome.4c00642

Shunsuke Takagi, Nobuyuki Suzuki, Yasushi Ishihama

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引用次数: 0

摘要

我们使用为生物制药（如单克隆抗体）开发的最先进的反相色谱柱重新研究了蛋白质反相色谱（RP），以评估这种方法是否适合作为自下而上蛋白质组学的预抽提步骤。以细胞裂解液为样品，将蛋白质 RP 预分馏（Prot-RP）方法与另外两种广泛使用的预分馏方法（SDS-PAGE 和高pH 肽 RP（Pept-RP））进行了比较。结果表明，Prot-RP 法各组分间的重叠度与 SDS-PAGE 法相当，蛋白质回收率高出约 2 倍。另一方面，与 Pept-RP 相比，Prot-RP 各组分间的重叠率稍高，但 Prot-RP 能鉴定出更多的蛋白质末端和更多的同工酶特异性肽段。我们的研究结果表明，将高效蛋白质预分馏与现代质谱仪相结合，对细胞样本的蛋白质形态分析特别有效。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics

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Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics

We revisited protein reversed-phase chromatography (RP), using state-of-the-art RP columns developed for biopharmaceuticals, such as monoclonal antibodies, in order to evaluate the suitability of this methodology as a prefractionation step for bottom-up proteomics. The protein RP prefractionation (Prot-RP) method was compared with two other widely used prefractionation methods, SDS-PAGE and high-pH peptide RP (Pept-RP) by using cell lysates as samples. The overlap between fractions of Prot-RP was comparable to that of SDS-PAGE, and the protein recovery was approximately 2-fold higher. On the other hand, the overlap between fractions of Prot-RP was slightly larger than that of Pept-RP, but Prot-RP was able to identify more protein termini and more isoform-specific peptides than Pept-RP. Our results indicate that the combination of highly efficient protein prefractionation with modern mass spectrometers is particularly effective for proteoform profiling from cellular samples.

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来源期刊

Journal of Proteome Research 生物-生化研究方法

CiteScore

9.00

自引率

4.50%

发文量

251

审稿时长

3 months

期刊介绍： Journal of Proteome Research publishes content encompassing all aspects of global protein analysis and function, including the dynamic aspects of genomics, spatio-temporal proteomics, metabonomics and metabolomics, clinical and agricultural proteomics, as well as advances in methodology including bioinformatics. The theme and emphasis is on a multidisciplinary approach to the life sciences through the synergy between the different types of "omics".