The liquid nature of the matrix used in this study makes the adjustment of physicochemical properties of the analyte–matrix
environment possible. Here we show that this novel liquid matrix system can be adjusted within the pH range of 3–8.75 when
diluted, leading to a pH range of 3.5–8.6 when dried on target without losing its liquid nature and high analytical sensitivity.
The pH of the diluted matrix without pH adjustment is ~5.0 (5.7 when solvents are evaporated). Several bases and buffers have
been investigated to assess their ability to alter the pH of the matrix while maintaining sensitivity and liquidity. In many
cases, crystallization occurred before a significant pH increase was observed; in others, low solubility in the dried (that
is, solvent-evaporated) matrix caused the formation of a two-phase liquid in which the matrix components separated during
solvent evaporation, generating two distinct overlapping droplets of differing color. So far, the best buffer found as tolerable
additive and effectively changing the pH is ammonium bicarbonate. A pH of 8.6 is achievable on target with an ammonium bicarbonate
concentration of 200 mM in the nondried diluted matrix solution without any signs of crystallization. Crystallization has
been observed at 300 mM. However, there is no significant pH increase due to the buffering nature of the ammonium bicarbonate.
The pH begins to reach a plateau at 50 mM (pH 8.3). To lower the pH, trifluoracetic acid was added. A pH of 3.9 (0.3% trifluoracetic
acid) has been achieved on target without any crystallization. At 0.35% trifluoracetic acid (pH 2.8 diluted matrix, 3.5 dried),
some very minor crystallization was observed, but this did not appear to affect the matrix performance. As the trifluoracetic
acid was increased further, more crystallization was observed, now severely reducing the matrix performance. Total crystallization
was obtained at 0.5% trifluoracetic acid. The current pH range achievable for the diluted and dried matrix can be seen in
Figure 4.
Figure 5
Peptides are zwitterionic, and the charge of a peptide in solution is determined by the pH of the solution. The solution pH
at which the peptide as a whole has no charge is called isoelectric point, or pI. The peptides in the analyte mixture used
have a range of pI values from 4.0 to 11.7. In Figure 5, the signal-to-noise ratios (S/N) of three of the peptides with a
wide range of pI values but similar masses have been compared (bradykinin with pI 9.7, angiotensin I with pI 6.9, and Glu-Fib
with pI 4.0). A graph similar to the one in Figure 5 was obtained for intensities. To compare the peptides, which are present
in varying concentrations, the values have been normalized against the mean value for the unadjusted matrix at pH 5. It can
be seen from Figure 5 that the pI does not significantly affect the ionization of peptides across a wide pH range with similar
values for each of the peptides across the entire range. Figure 6 shows the overall performance of the matrix across the pH
range. The normalized values for all six peptide peaks were averaged for the intensity and S/N achievable. Despite some minor
fluctuations, the majority of values fall on the 100% line. Although the pH of the matrix does not appear to affect the ionization
of these peptides, it might be important for other classes of peptides or biomolecules such as phosphopeptides or DNA and,
in general, for acid- or base-labile compounds. These classes of compounds will be investigated in the future.
