AND MASS SPECTROMETRY 5
11. MONOLITHIC LAYERS FOR THIN-LAYER CHROMATOGRAPHY e MASS SPECTROMETRYSPECTROMETRY
11.1 THIN-LAYER CHROMATOGRAPHY SEPARATION AND MASS SPECTROMETRIC DETECTION OF BIOMOLECULESDETECTION OF BIOMOLECULES
that desorption/ionization in MALDI occurs only on the surface, not deeper than a couple of hundreds of nanometer. Because the compound is distributed throughout the entire thickness of the layer, its amount in the thin surface film is small. The presence of the less permeable “skin” at the top of the layer, which is permeable only through the cracks, also contributes to the loss of both sensitivity and repeatability. Although the use of a monolithic layer in 2D separations of proteins remains vital, similar studies should be repeated with a more precise porous polymer substrate to avoid the undesired interferences of thickness and the “skin.”
11. MONOLITHIC LAYERS FOR THIN-LAYER CHROMATOGRAPHY e MASS
types of layers was desirable. The launch of monolithic plain silica-based ultrathin layers was one of the answers to this challenge. However, production of these layers was short-lived (vide supra).
We showed earlier in this chapter that preparation of thin layers supported by glass slides is simple.
Thus, a 50-mm-thick monolithic poly(butyl methacrylateeethylene dimethacrylate) layer was pre- pared and used for separation of fluorescamine-labeled peptides and proteins (Bakry et al., 2007). The labeling was required to observe the separated spots. Because the direct ionization of peptides from the monolithic layer was poor, CHCA was applied to amplify ionization, leading to a significant enhancement in ionization efficiency. Both spraying and spotting protocols were tested for matrix application. On spotting, undesired lateral migration of compounds in the thin layer was observed after the droplet of matrix solution was applied. This migration then moved the spot away from the original location and led to a decrease in resolution. Therefore, spraying was applied throughout the entire study. The enhancement in ionization efficiency likely resulted from the extraction function of the matrix solution, which increased the concentration of the peptide at the top of the monolithic layer, as suggested byGusev et al. (1995b).
Fig. 5.13shows the separation of three peptides and their characterization in spots using MALDI MS. Migration to the distance of 6 cm was rather fast and required only 5e6 min using 0.1 vol%
trifluoroacetic acid in 40 vol% aqueous acetonitrile as the mobile phase. Spectra of [Sar1,Ile8]- angiotensin II, angiotensin II, and neurotensin feature two peaks, indicating that the spots contained both the original peptides and their fluorescamine-labeled counterparts (Bakry et al., 2007).
Similarly, proteins were also separated. A short optimization revealed that 0.1 vol% trifluoroacetic acid in 55e60 vol% aqueous acetonitrile was the most efficient mobile phase for that separation.
Fluorescent labeling of the proteins enabled scanning of the plate using a fluorodensitometer, after separation of the spotted solution containing 1 pmol/mL of each compound. However, the labeling produced a host of biomolecules varying in the number of attached fluorescamines, which complicated quantification. To avoid this problem, nonlabeled proteins were detected. The sampling point was marked at the plate and scanning proceeded along the developed line.Fig. 5.14shows the MALDI spectra of each separated protein acquired using sinapinic acid as the matrix because this compound was considered to be the matrix of choice for proteins over a wide mass range.
The previous study also included the preparation of a monolithic layer from a mixture consisting of styrene, divinylbenzene, 1-decanol, and tetrahydrofuran, with azobisisobutyronitrile as an initiator.
Motivation for the use of this polymer was the assumption that the aromatic character of the layer might affect hydrophobicity and also contributes to the selectivity through thepep interactions.
Because these monomers are UV absorbing, the UV light is absorbed by the monomers and does not reach the initiator to achieve its decomposition to free radicals. Therefore, polymerization required thermal initiation. The poly(styrene-co-divinylbenzene) layers prepared this way were poor in quality and several defects such as cracks and nonuniform thickness could be observed. Therefore, this polymer layer was not used in the above project and more experiments were needed to obtain a useful thin layer.
The following experiments revealed that the major problem was the “skin” containing cracks and pinholes located at the external surface of the layer, such as those that were shown in Fig. 5.10.
Removal of this “skin,” as described above, and optimization of the composition of the polymerization mixture solved the problem (Lv et al., 2013).
The polymerization mixture used for the preparation of the layer was comprised of the monomers 4-methylstyrene, chloromethylstyrene, and divinylbenzene, porogens toluene and 1-dodecanol, and
azobisisobutyronitrile initiator. An SEM image of the layer is presented inFig. 5.10. Compared with the previous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) plates presented by Bakry et al., the styrenics-based monolithic layers exhibited higher hydrophobicity, which was confirmed by better separation and slower migration of peptides, even in the mobile phase containing a higher percentage of acetonitrile. It is worth noting that the preparation is highly reproducible.
FIGURE 5.13
Thin-layer chromatography separation of mixture of peptides labeled with fluorescamine using poly(butyl acrylate-co-ethylene dimethacrylate) monolithic layer attached to a glass plate using 0.1 vol% trifluoroacetic acid in 45 vol% aqueous acetonitrile as the mobile phase (left) and matrix-assisted laser desorption/ionization time-of- flight mass spectrometryspectra of fluorescently labeled [Sar1,Ile8]-angiotensin II, angiotensin II, and neurotensin obtained “from-plate” usinga-cyano-4-hydroxycinnamic acid as matrix.
Experiments with eight different plates and double spotting of peptide mixtures demonstrated excellent repeatability with an RSD forRfof less than 2.5%.
This layer was also used for the separation of three fluorescamine-labeled proteins. Ribonuclease A, lysozyme, and myoglobin were separated in 15 min to distinct spots shown inFig. 5.15. The elution order followed the hydrophobicity of the individual proteins, and MALDI mass spectra confirmed the FIGURE 5.14
Matrix assisted laser desorption/ionization spectra of nonlabeled cytochrome c (1), lysozyme (2), and myoglobin (3) separated on 50-mm-thick poly(butyl acrylate-co-ethylene dimethacrylate) monolithic layer attached to a glass plate using 0.1 vol% trifluoroacetic acid in 60 vol% aqueous acetonitrile as the mobile phase obtained “from- plate” using sinapic acid as matrix.
FIGURE 5.15
Thin-layer chromatography separation of a mixture of proteins ribonuclease A, lysozyme, myoglobin, and myoglobin dimer labeled with fluorescamine using 50-mm-thick poly(4-methylstyrene-co-chloromethylstyrene- co-divinylbenzene) monolithic layer developed by 65% acetonitrile in 0.1% aqueous trifluoroacetic acid solution and matrix-assisted laser desorption/ionization time-of flight mass spectrometry spectra obtained from the spots using sinapic acid matrix.
identity of the labeled proteins. The fourth spot with anm/z¼34841.579, which had the smallestRf, was assigned to a myoglobin dimer, which is a known impurity in the commercial protein. Although the MS signal of this peak was weak because of the low concentration of the dimer in the sample, it contained two fluorescamine labels that enhanced the fluorescence and enabled visualization of its spot. The repeatability ofRfwas again excellent with an RSD of less than 2.4%.
An additional benefit of the chemistry of these thin layers lies in their ability to get hyper- crosslinked. The FriedeleCrafts alkylation reaction catalyzed by ferric chloride is known to form a large number of mesopores and leads to a significant increase in the surface area. Hypercrosslinking of the monoliths containing chloromethylstyrene units was introduced for monolithic capillary columns only recently (Maya and Svec, 2014; Skerı´kova´ and Urban, 2013; Urban et al., 2010a,b;
Urban and Skerı´kova´, 2014) and so has not been used previously for thin layers. In contrast to the original plate, a hypercrosslinked layer was able to separate even small molecules such as dyes (Lv et al., 2013).