Low flow liquid chromatography-native mass spectrometry for the analysis of proteoforms and complexes

The thesis investigates the characterization of intact proteins, proteoforms, and protein complexes from the native perspectives. Traditionally, the peptide-centric methods (e.g., bottom-up methods) are the main strategy to identify proteins. However, the digestion procedures and/or denaturing environments usually result in protein cleavage and unfolding, leading to critical information loss (e.g., non-covalent interactions). To overcome these drawbacks, this research focuses on developing low-flow native separation systems hyphenated with native mass spectrometry (nMS) to assist the comprehensive identification of their size, chemical composition, conformation, and heterogeneity. Chapter 2 compared native microflow size-exclusion chromatography (SEC)-nMS and denaturing nanoflow hydrophilic-interaction chromatography (HILIC)-MS for studying glycated proteins, proving that the native condition can provide more information on the aggregates. From microscale to nanoscale, Chapter 3 develops a nanoflow SEC-nMS method for studying various proteins and their complexes, in which a milder condition and higher sensitivity are achieved. Compared with SEC, ion-exchange chromatography (IEC) has great advantages in differentiating proteoforms. Chapter 4 summarizes the recent development of IEC-MS for intact protein analysis. Based on it, the nanoflow IEC-nMS was developed in Chapter 5, allowing for separating large proteoforms with minor mass difference in a non-denaturing condition. To further increase the separation of complicated mixtures, Chapter 6 created a double-barrel setup by integrating nanoflow cation- and anion-exchange modes together, with which the acidic and basic proteoforms can be simultaneously resolved in a time-saving and high-throughput way. The developed native low-flow LC-nMS platform opens unprecedented opportunities for clinical studies and other sample-limited applications.