Matrix assisted laser desorption ionization (MALDI) provides sufficiently mild conditions to transfer entire pepsiRNAs into the gas phase. Commercially obtained thiol-labeled siRNAs were reacted with biotin-labeled activated PenetratinTM in an equimolar ratio for one hour. After gel filtration, the reaction mixture was prepared for MALDI-MS. Trihydroxyacetophenone (THAP) has favorable properties as a matrix for the measurement of oligonucleotides (Jensen et al. 1996). First measurements were carried out in the negative mode with an DNA-oligonucleotide of known size as a reference.
Figure 3-18 MALDI analysis of pepsiRNAs a) The MALDI spectrum of the unmodified substance (negative mode, matrix: THAP, NH4Ac added) shows five peaks corresponding to ssRNA (~7kDa),
ssRNA coupled to PenetratinTM (~9.8 kDa), siRNA (~14kDa) and siRNA coupled to PenetratinTM [M]-- ~16.8 kD, [M]2-~8.4 kDa).
b) Denaturation of the sample by heating to 80°C and immediate cooling to 0°C before preparation leads to a significant reduction of the peaks representing dsRNA.
c) Peaks corresponding to disulfide-coupled compounds are diminished if the compound is treated with DTT to reduce disulfide bonds prior to sample preparation. (calculated masses: sense-RNA 6.804 kDa, asRNA 6.757kDa, ssRNA-PenetratinTM-biotin 9.379, siRNA 13.561 kD, siRNA-PenetratinTM-biotin 16.137kD).
a)
b)
Both pepsiRNA and standard oligonucleotide were detected as broadened peaks. Due to the high acidity of the backbone phosphates, these residues are deprotonated at neutral pH. The siRNAs bear 42 negatively charged residues and are thereby found as mixtures of sodium and potassium salts, so that broadened peaks are found. As described by Jensen (Jensen et al. 1996), the pepsiRNA and reference samples were treated with ammonium acetate to exchange sodium and potassium ions for ammonium ions that dissociate to volatile NH3 and
H+ in the gas phase leaving behind protonated phosphate residues. The thus obtained peaks were much more focused, but could still not be assigned to one distinct mass. In three measurements of aliquots of the same sample, the value for the dsRNA peak varied between 14180 and 14649 Da corresponding to a maximum deviation of 1.8% from the average of the three measured values. This was much higher than the deviations of 0.08% reported for short model conjugates of peptides with oligodeoxynucleotides of an overall mass of 1942 to 4381 Da as determined by MALDI-MS (Jensen et al. 1996). One reason for this was the low signal to noise ratio obtained for the pepsiRNAs, which led to ambiguous results in the determination of the peak maximum. Additionally, the incomplete removal of associated Na+- ions resulted in a mass distribution of the oligoribonucleotides, and finally, the siRNAs might have been subject to nuclease degradation during sample preparation leading to fragments of different size. Evidence for this comes from 12% TBE-PAGE (7 M urea) by which fragments could be detected in a sample of radioactively labeled, denatured siRNAs (see Figure 3-19). This could be also due to strand termination during solid phase synthesis.
Figure 3-19 12% TBE-PAGE (7 M urea) with three different aliquots of a commercially obtained siRNAs to target HexA. siRNAs were labeled at their 5´-ends with [32P]-γ-ATP by T-4 kinase, dissolved in loading buffer and denatured prior to loading to the gel. Radioactivity was detected with a phosphoimager screen (Bas III, Fuji). The siRNAs do not exhibit a uniform size distribution. Shorter nucleotides may result from degradation by nucleases during sample preparation or from strand termination during solid phase synthesis.
In the mass sprectrum obtained for the pepsiRNA sample, all expected species are present: pepsiRNAs at ~16.8 kDa and ~8.5 kDa (for the doubly charged species), free siRNAs at ~14kDa, Penetratin coupled to the siRNA sense strand at ~9,8 kDa and 21nt single stranded RNAs that form the largest peak at ~7 kDa. The chemical identity of these peaks was confirmed by two experiments as shown in Figure 3-18.
Denaturation of the sample by heating to 80°C and rapid cooling on ice greatly reduced the amount of double stranded RNA (14 kDa) and pepsiRNAs (16.8 kDa). Reduction of the sample with DTT prior to measurements led to the elimination of the peaks representing the disulfide-bridged compounds (double stranded (16.8 kDa) and single stranded RNA- penetratin conjugates (9.8 kDa).
The peak corresponding to single stranded RNAs is large compared to dsRNA peptide coupled RNAs. It has been reported, that conjugates of peptides and nucleotides pose special problems, since the two polymeric components have conflicting requirements for ionization (Jensen, 1996). This may be more pronounced for the larger conjugates as 21 bp siRNA coupled to the 16 residues of PenetratinTM, than for the reported conjugates of an 11- amino-acid peptide with thymidine decamers and hexamers. It is also likely that a proportion of the dsRNAs dissociated during the ionization process or that the double strands partially separate in the desalted solution after gel filtration. The signal at 7.2 or 7.0 kDa originated from dissociated dsRNAs as well as from antisense-strands dissociated from the pepsiRNA. However, the existence of a dsRNA signal indicated, that not all of the siRNAs in the reaction mixture had reacted with the peptide.
Thus, the result from the MALDI-TOF-MS analysis has to be evaluated as proof of the existence of peptide-coupled siRNAs. It cannot be taken as a quantitative measure of the coupling efficiency, but indicates qualitatively that not all of the siRNAs were coupled to the peptide, which could not be deteced by SDS-PAGE. Therefore, for the following cell culture studies one must consider that the concentration estimated for the experiments might be much lower than depicted, even though the pepsiRNAs were subjected to gel-filtration. The final concentration was determined by photometrical measurements at 260 nm.