Hollow‐Fiber Flow Field‐Flow Fractionation: A Gentle Separation Method for Mass Spectrometry of Native Proteins

Abstract

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

“…In native MS, where physiological or near-physiological conditions are required, the task of separating non-denatured proteins and protein complexes at high resolution is further complicated. Conventional separation techniques compatible with native MS include size exclusion chromatography (SEC) [18], ion exchange chromatography (IEX) [19], hydrophobic interaction chromatography (HIC) [20], affinity chromatography [21], capillary isoelectric focusing (cIEF) [22, 23], native gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) [24, 25], native polyacrylamide gel electrophoresis [26], capillary zone electrophoresis (CZE) [27, 28] and flow field-flow fractionation (F4) [29, 30]. However, for many of these methods, online interfacing to a mass spectrometer is challenging or yet impossible.…”

Standard procedures for native CZE‐MS of proteins and protein complexes up to 800 kDa

“…"Native-friendly" chromatographically based separation approaches include size exclusion chromatography [27], hydrophobic interaction chromatography [28], ion-exchange chromatography [29], and affinity chromatography [30]. In addition, electromigrative separation techniques such as capillary isoelectric focusing [31], native Gel-Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) [32], flow field-flow fractionation [33], and CZE [34,35] have been applied for native protein analysis. In this context, three major prerequisites must be fulfilled to achieve efficient native separation coupled to an MS instrument: (i) the buffer/electrolytes must preserve native features of the protein or protein assembly; (ii) the composition of the spray solution must be compatible with electrospray ionization to avoid ion suppression; and (iii) the separation performance and resolution need to be sufficient to separate targeted analytes.…”

Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis–Top-Down Mass Spectrometry

“…Different analytical chromatographic − and electromigrative − tools theoretically allow native separation. Generally, there are three major requirements to achieve efficient native separation prior to MS detection: (i) the mobile phase or background electrolyte (BGE) must maintain the nature of the nondenatured protein or complex, (ii) the composition of the mobile phase/BGE must be compatible with electrospray ionization, and (iii) the separation performance and resolution need to be sufficient.…”