study competition between adsorption of insulin to nitrocellulose and self ... Adsorption of proteins to nitrocellulose has been demonstrated to be very useful as a ...
JOURNAL DE PHYSIQUE Colloque C2, suppl6ment au n02, Tome 50, fgvrier 1989
PLASMA DESORPTION MASS SPECTROMETRY STUDIES OF SMALL PROTEINS ADSORBED TO NITROCELLULOSE G. JONSSON, A. HEDIN, P.
HAKANSSON
and B.U.R.
SUNDQVIST
Division of Ion physics, Department of Radiation Sciences, Uppsala University, Box 535, S-751 21 Uppsala, Sweden
B,&um4-L'adsorption d'insuline bovine sur de la nitrocelluiese a 6th ktudi6e par spectrom6trie de masse PDMS et par ellipsomktrie. Les rksultats montrent que la spectromktrie de masse PDMS peut 6tre utiliske pour l'btude de la compktition entre l'adsorption directe d'insuline sur la nitrocellulose et la formation d'agglomkrats d'insuline.
Abstract-Adsorption of bovine insulin to nitrocellulosc has been studied using Plasma Desorption Mass Spectrometry (PDMS) and ellipsometry. The results indicate that PDMS can be used to study competition between adsorption of insulin to nitrocellulose and self association.
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1 INTRODUCTION
Adsorption of proteins to nitrocellulose has been demonstrated to be very useful as a sample preparation method in Plasma Desorption Mass Spectrometry (PDMs) (1). In this study three experiments have been performed with the aim to learn more about the application of nitrocellulose for sample preparation in PDMS.
In a previous study (2), nitrocellulose was electrosprayed onto smooth silicon backings. Small proteins, e.g. bovine insulin (MW 5733), dissolved in 50 % of 0.1 % TFA-solution (1 I TFA-solution: 1 ml trifluoro acetic acid1999 ml HzO) and 50 % of 95 % ethanol, were adsorbed onto the nitrocellulose surface and the sample surface was then washed with 0.1 % TFA-solution (3). The yields of (M+H)+ and (M+2H)'+ were measured with PDMS for different amounts of insulin applied onto the nitrocellulose surface (figure 1 in ref. 2). In the other two studies, homogeneous films of nitrocellulose, 30-800 A thick, were spin-coated (4) onto silicon. Film thicknesses were measured with an ellipsometer. In the first of these two studies, the amount of bovine insulin deposited onto the surface was kept constant (2nmole) and the film thickness was varied, whereas in the latter study, the film thickness was kept constant (about 450 A) and the amount of protein applied,was varied. The same solvent was used for the sample molecules as in the former experiment. In both cases, the surfaces were then washed with pure water after the adsorption. In the last experiment the total insulator thickness was measured after rinsing. The thickness difference was taken as a measure of the thickness of the insulin layer after washing. Finally, the PDMS-yield for (M+II)+ and (M+2H)2+ was measured as a function of nitrocellulose film thickness and amount of bovine insulin left on the surface.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1989211
JOURNAL DE PHYSIQUE
3
- RESULTS AND DISCUSSION
In the previous study (2), in which electrosprayed nitrocellulose was used, the yield ratio (M+H)+ to (M+2H)2f was found to increase with increasing amount of insulin applied (figure 1 in ref. 2). This might be interpreted as a competition between self association and adsorption to nitrocellulose (2). Results from experiments on other proteins show the same trend, but the effect is most pronounced for insulin of the molecules studied so far. This is in agreement with the fact that insulin is known to cluster easily (5). In the second study, the yield for bovine insulin was found to increase with film thickness up to about 200 A and then saturate (cf. figure 1, the dashed line is a guide to the eye). In a study by SZve et at. (4), it was found that the yield of protonated bovine insulin from a spin-coated layer saturates at about the same film thickness. These data (4) have been interpreted as mainly due to crater formation (6): However, the present experiment described here indicates that an insulating layer of a certain thickness is needed for efficient electronic sputtering. The underlaying locally expanding nitrocellulose may help to eject the insulin molecules. It is also interesting to note that the yield for these spin-coated backings is almost as high as for the electrosprayed nitrocellulose backings, in spite of the much larger active area for the latter backings. Maybe, the insulin is not evenly distributed over the inhomogeneous electrosprayed nitrocellulose surface. The results of the third study are similar to the results in the first study. In figure 2 spectra of molecular ions of bovine insulin for different amounts of sample applied are shown. Preliminary measurements with ellipsometry of the amount of insulin adsorbed to the nitrocellulose surface after rinsing. indicate that monolayer coverage in this experiment corresponds to 1-4 nmoles applied. The results in figure 2 therefore illustrates that the relative intensity of molecular ions changes below monolayer coverage. It therefore seems that PDMS can be used to probe the competition between adsorption of insulin to nitrocellulose and self association. The fact that the insulins associate will of course disturb the ellipsometry measurement as the accuracy of that method is dependent on the formation of a homogeneous film. Still the identification of a region of applied amount corresponding to monolayer coverage is probably reliable.
Figure 1.
THICKNESS
(A)
Figure 2.
1
l0O1
0.1nmole
I
I
0.01 nmole
REFERENCES 1) .TONSSON G., HEDIN A., HAKANSSON P., SUNDQVIST B. U. R., SAVE G . , NIELSEN P., ROEPSTORPF P., JOHANSSON K.-E., KAMENSKY I. and LINDRERG M., (1986) Anal. Chem., 58, 1084 2) JONSSON G., HEDIN A., HAKANSSON P. and SUNDQVIST B. U. R., (1988) Rap. Comm. Mass Spectrom., 2, 154 3) ROEPSTORPF P., NIELSEN P., SUNDQVIST B. U. R., HAKANSSON P. and JONSSON C . , (1987) Int. J. Mass Spectrom. Ion Proc., 78, 229 4) SAVE G., HAKANSSON P., SUNDQVIST B. U. R. and JONSSON U., (1987) Nucl. Instr. Meth., B 2 6 , 571 5) BLUNDELL T., DODSON G., HODGKIN D. and MERCOLA D., (1972) Adv. Protein Chem., 26, 279 6 ) SAVE G., HAKANSSON P., SUNDQVIST B. U. R., SO~>ERSTROME., LTNDQVTST S.-E. and BERG J., (1057) Appl. Phys. Lett., 51, 1379
