Revisiting\nProtein Reversed-Phase Chromatography for\nBottom-Up Proteomics

We revisited protein reversed-phase chromatography (RP),\nusing\nstate-of-the-art RP columns developed for biopharmaceuticals, such\nas monoclonal antibodies, in order to evaluate the suitability of\nthis methodology as a prefractionation step for bottom-up proteomics.\nThe protein RP prefractionation (Prot-RP) method was compared with\ntwo other widely used prefractionation methods, SDS-PAGE and high-pH\npeptide RP (Pept-RP) by using cell lysates as samples. The overlap\nbetween fractions of Prot-RP was comparable to that of SDS-PAGE, and\nthe protein recovery was approximately 2-fold higher. On the other\nhand, the overlap between fractions of Prot-RP was slightly larger\nthan that of Pept-RP, but Prot-RP was able to identify more protein\ntermini and more isoform-specific peptides than Pept-RP. Our results\nindicate that the combination of highly efficient protein prefractionation\nwith modern mass spectrometers is particularly effective for proteoform\nprofiling from cellular samples.