AND MASS SPECTROMETRY 5
10. MONOLITHIC LAYERS ENHANCING DESORPTION/IONIZATION
orders of magnitude, implying that no significant interferences are to be observed under normal use of the monolithic porous polymer matrix.
The pore size of the monoliths also plays a role in desorption and ionization. Fortunately, the easy control of porous properties via variation of the composition of the polymerization mixture is another advantage of the porous polymer monoliths, and enables a facile optimization of the pore size for the desired application. An effect of the solvent used for sample preparation was also observed. This is likely due to the differences in the wetting of the monolithic surface.
In contrast to many desorption/ionization matrices, exposure of the porous polymer monolith to air is not a problem because it does not contain any oxygen sensitive functionalities at the surface that would cause a loss of performance. Fig. 5.12 compares the mass spectrum of caffeine recorded immediately after spotting the monolithic layer to that obtained using the same sample on the same spots on a plate that had been left unprotected in the laboratory for 3 weeks. The spectra are practically identical. This demonstrates that both the monolithic matrix and the sample contained in the plate did not change even after a long period of time, and no specific precautions have to be applied to avoid contact with the environment. This finding also suggests that the monolithic spots can be used for the storage and archiving of at least nonvolatile samples.
One more advantage of the monolithic plates lies in their neutral chemistry. It is well known that many conventional low-molecular weight-matrices used in MALDI have an acidic character. This makes the use of MALDI difficult for the analysis of acid-labile compounds because they may decompose before being desorbed and ionized. For example, a complex, acid sensitive N,Nʹ- bistrifluoroacetyl-di-(2-aminoethoxy)-[4-(1,4,7,10-tetraoxaundecyl) phenyl] methane with a molecu- lar mass of 564 did not afford a useful MALDI-TOF MS mass spectrum using any common low molecular weight matrices. In contrast, ionization from the monolithic layer afforded a good spectrum that included peaks for the molecular ion and the sodium and potassium adducts (Peterson et al., 2004).
This result clearly demonstrated that monolithic plates and spots can facilitate the ionization of numerous compounds that would be very difficult or impossible to analyze using more conventional MALDI techniques and matrices.
Instead of monolithic cylinders, Wouters et al. used photopolymerized 100mm thick continuous monolithic poly(butyl methacrylateeethylene dimethacrylate) layers. The only difference consisted in FIGURE 5.11
Laser desorption/ionization mass spectra of blank spot of porous butyl methacrylateebased monolith and of 2,5- dihydroxybenzoic acid matrix.
application of a different porogenic system comprising 1,4-butanediol and 1-propanol. Motivation for their study was testing the potential of the monolith as a replacement for the labor-intensive and slow 2D polyacrylamide gel electrophoresis (PAGE) technique typically used in proteomics. They spotted solutions of the peptide angiotensin and protein cytochrome c on the monolithic layer while applying the same visualization methods as in PAGE, i.e., silver nitrate and Coomassie brilliant blue R to visualize and quantify. The detection limits were found comparable to those found for PAGE; however, the staining was achieved in a mere 10e15 min. In addition, fluorescamine-labeled angiotensin and cytochrome c were also spotted and amounts down to at least 6 ng could be detected. Complaints of the authors related to the poor spot-to-spot repeatability are likely the result of the heterogeneity of the surface “skin” discussed above.
The peptide and protein spots at the monolithic layer were covered with a solution of MALDI matrix CHCA, and mass accuracy, mass precision, and signal intensity were determined. Both angiotensin and cytochrome c were also spotted together with CHCA on the standard stainless steel MALDI plate. Based on results of the measurements, the authors concluded that the monolithic layers are unsuitable for the top-down identification of proteins, based on comparison with the direct spotting on the plate. However, there might be several reasons for the unsatisfactory results, with some of them mentioned by the authors themselves. Certainly, a thickness of 100mm is too large, taking into account
100 0 3000 6000 25000
Intensity (arb.units)
109 137
O
O N N
N
Caffeine
195
217
Immediately after spotting
N 50000
109
137
195
217 Three weeks later
150 200
m/z
250
FIGURE 5.12
Mass spectrum of caffeine desorbed/ionized from surface of porous butyl methacrylateebased monolith obtained immediately after preparation of the spot and spectrum of the same spot recorded 3 weeks after the previous one.
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.”