The Potential of Polymeric Monolithic Capillary Columns for the LC–MS Analysis of Intact Proteins - - Chromatography Online
The Potential of Polymeric Monolithic Capillary Columns for the LC–MS Analysis of Intact Proteins

LCGC Europe
Volume 25, Issue 1, pp. 10-19

LC–MS-based top-down proteomics involves the chromatographic separation and mass-spectrometric characterization of intact proteins without prior enzymatic cleavage into peptides. This article describes the preparation of monolithic reversed-phase (RP) columns with optimized porous structure and their performance separating intact proteins. Peak capacities of around 600 could be achieved within a 2 h gradient. The combination of high-resolution monolith chromatography with high-accuracy timeofflight mass spectrometry (TOF-MS) allowed intact proteins and protein isoforms that differ only in their oxidation state to be distinguished. The article also describes various MS fragmentation techniques for the identification and the more detailed characterization of intact proteins.

The concept of separation media based on monolithic materials can be traced back to the early 1950s, when Nobel Prize Laureates Martin and Synge discussed the benefits of these materials for different applications in chromatography (1). One of the first monolithic polymer gels for liquid chromatography (LC) was developed by Kubín et al. in 1967 (2). Highly swollen hydrogels, prepared in glass tubes from 2-hydroxyethyl methacrylate as the bulk monomer and ethylene dimethacrylate as the cross-linking monomer, were applied for the size-exclusion separations of water-soluble polymers. Rigid macroporous polymer-based monolithic columns were introduced in the early 1990s (3). This type of stationary phase is composed of a single monolithic unit with interconnected microglobules and macropores (flow-through pores), and is typically bonded to the column wall via a covalent bonding to enhance the robustness.

Polymer monoliths are prepared from liquid precursors, that is, the bulk monomer and cross-linker dissolved in the porogen, allowing their in-situ preparation in virtually any format. The separation performance strongly depends on the porous structure of the monolithic material (4). It has been demonstrated that the composition of the polymerization mixture, including the type or ratio of the porogen solvents in the polymerization mixture (5), and the polymerization conditions, such as polymerization temperature and time (6), are key parameters that need to be controlled precisely. The surface chemistry and therefore selectivity can be tuned by incorporating 'functional' monomers in the polymer backbone (7). As a result of the absence of mesopores (stagnant zones inside polymer microglobules), the mass-transfer contribution to total band broadening is greatly reduced (8).

The success of polymer monolithic columns for the reversed-phase gradient-elution separation of peptides in a LC–MS bottom-up proteomics approach has been demonstrated on several occasions (9–11). Karger et al. explored the use of 20 µm i.d. monolithic columns and established an HPLC–ESI–MS method yielding low-attomol detection sensitivity (12). Recently, we demonstrated a LC–MS–MS separation of a tryptic E. coli digest on a 1 m monolithic column yielding a peak capacity in excess of 1000 (13).

LC–MS-based top-down proteomics is based on the chromatographic separation of intact proteins followed by their mass spectrometric elucidation. Single-stage MS provides information on the molar mass of intact proteins. The mass alone can give information about chemical or post-translational modifications of proteins, provided that the identity of the protein is already known. Tandem mass spectrometry MSn can provide structural information and is essential for the identification of unknown proteins. In MSn the selected precursor ions are fragmented in the gas phase using neutral gas molecules for collision-induced dissociation (CID) (14) and higher-energy collisional dissociation (HCD) (15); electrons for electron capture dissociation (ECD) (16); or radical anions for electron transfer dissociation (ETD) (17). In top-down proteomics high-resolution separation technology is of vital importance to address two bottlenecks: ion suppression and the spectral complexity.

Here, the potential of capillary polymer monolithic columns is discussed for the separation of intact proteins, including protein isoforms. The effects of column properties (length and morphology) and gradient conditions on the separation performance were evaluated. The potential to achieve high-resolution protein separation is demonstrated with the liquid chromatography time-of-flight mass spectrometry (LC–TOF-MS) analysis of a mixture containing 48 intact human proteins. In addition, the application of complementary fragmentation techniques (CID, HCD and ETD) is demonstrated for protein identification using a hybrid ion trap-orbitrap mass spectrometer.


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