Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Analytical and Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd., Hiratsuka, Kanagawa 254-0014, Japan.
J Proteome Res. 2024 Oct 4;23(10):4704-4714. doi: 10.1021/acs.jproteome.4c00642. Epub 2024 Sep 18.
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.
我们重新研究了蛋白质反相色谱（RP），使用了专为生物制药（如单克隆抗体）开发的最先进的 RP 柱，以评估该方法作为用于自下而上蛋白质组学的预分级步骤的适用性。通过使用细胞裂解物作为样品，将蛋白质 RP 预分级（Prot-RP）方法与另外两种广泛使用的预分级方法 SDS-PAGE 和高 pH 肽 RP（Pept-RP）进行了比较。Prot-RP 馏分之间的重叠与 SDS-PAGE 相当，而蛋白质回收率约高 2 倍。另一方面，Prot-RP 馏分之间的重叠略大于 Pept-RP，但 Prot-RP 能够鉴定出比 Pept-RP 更多的蛋白末端和更多的同工型特异性肽。我们的结果表明，将高效的蛋白质预分级与现代质谱仪相结合，对于从细胞样品中进行蛋白质亚型分析特别有效。
