Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry

Abstract

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex's architecture and subunits' identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for topdown MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody−antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.

Microfluidic devices for protein analysis using intact and top‐down mass spectrometry

“…Efficient mixing is essential for not only the speed of analysis but also the feasibility of performing time-resolved reactions with accurate duration control and high time resolution, especially at low time scales. 55 The advantage of online mixing over offline mixing is that the reaction time is equivalent to the effective mixing time, which can be precisely regulated via adjustment of channel volume and flow rate, without interference from the duration of sample injection or data acquisition. In contrast, in an offline mixing approach (either automated or manual), the reaction proceeds until the detection step, making it difficult to accurately control the effective reaction time at will.…”

“…Integrating a finger-tight union on the chip provides a solution to make the emitter both replaceable and reusable. 55 It also allows flexible selection of proper emitters.…”

“…In addition to the low flow rate as mentioned in the Introduction section, the desolvation and ionization efficiencies needed for TD‐MS favor an ESI emitter with a small inner diameter (I.D.) of orifice 55 . The existing designs of ESI interfaces can be categorized into three types (Figure 2A).…”

“…Connecting the chip with a separate ESI source results in a large void volume. Integrating a finger‐tight union on the chip provides a solution to make the emitter both replaceable and reusable 55 . It also allows flexible selection of proper emitters.…”

“…47 So far, the functions integrated into microchips for MS analysis include sample preparation, 43,45 separation, 48,49 digestion, 50 preconcentration, 49 and desalting. 51 In particular, a number of microchip-based devices and strategies for intact protein analysis have emerged [52][53][54][55] (for an overview of applications of these microchips, see Figure 1). Here, we attempt to review the designs and fabrication approaches of reported microchips developed for TD-MS and summarize the design considerations that may inspire the development of microchips with better performance and wider applications.…”