We have recently demonstrated the ability of a C18 stationary phase with a positively charged surface (PCS-C18) to provide superior chromatographic separation of peptides using mobile phase acidified with a mere 0.01 % formic acid, significantly improving MS sensitivity. Here, we examined three columns packed with different PCS-C18 phases using the MS-favorable mobile phase acidified with low formic acid concentrations to establish the impact of separation performance and better MS sensitivity on peptide identifications. The surface charge interaction was evaluated using the retention of nitrate. The highest interaction was observed for the AdvanceBio Peptide Plus column. A surface charge-dependent shift in the retention time of peptides was confirmed with a change in formic acid concentration in the mobile phase. The separation performance of the columns with MS-favorable mobile phase acidified with low concentrations of formic acid was evaluated using well-characterized peptides. The loading capacity was assessed using a basic peptide with three lysine residues. Good chromatographic peak shapes and high loading capacity were observed for the Acquity Premier CSH C18 column, even when using a mobile phase acidified with 0.01 % formic acid. The extent of improvement in peptide identification when using reduced formic acid concentration was evaluated by analyzing the tryptic digest of trastuzumab and tryptic digest of whole bacteria cell lysate. Each column provided improved MS signal intensity and peptide identification when using the mobile phase with 0.01 % formic acid. The ability of the Acquity Premier CSH C18 column to provide better separation of peptides, even with a reduced formic acid concentration in the mobile phase, boosted MS signal intensity by 65 % and increased the number of identified peptides from the bacterial sample by 19 %. Our study confirms that significant improvement in the proteomic outputs can be achieved without additional costs only by tailoring the chemistry of the stationary phase to the composition of the mobile phase. Our results can help researchers understand the retention mechanism of peptides on the PCS-C18 stationary phases using low-ionic strength mobile phases and, more importantly, select the best-suited stationary phases for their LC-MS proteomic applications.
- Klíčová slova
- Formic acid, Peptide separation, Proteomics, Reversed-phase liquid chromatography, Stationary phases,
- MeSH
- chromatografie kapalinová metody MeSH
- formiáty * chemie MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- peptidy chemie analýza izolace a purifikace MeSH
- proteomika * metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trastuzumab chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- formiáty * MeSH
- formic acid MeSH Prohlížeč
- peptidy MeSH
- trastuzumab MeSH
Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.
- MeSH
- alkylace MeSH
- footprinting proteinů metody MeSH
- halogenace MeSH
- imunokomplex chemie MeSH
- lidé MeSH
- mapování epitopu * metody MeSH
- monoklonální protilátky chemie imunologie MeSH
- receptor erbB-2 * chemie imunologie MeSH
- trastuzumab * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
