Comparison of MALDI Sample Preparations

Seven different MALDI sample preparations were tested for reproducibility and sensitivity. The sonicated dried droplet method was based on Howard and Boyer’s research.¹⁸⁴ Traditionally, this sample preparation may have been performed by mixing the two solutions directly on the target with an auto-pipette (a dried droplet preparation). Three thin-layer preparations were also assessed. These were prepared on a Bruker anchorchip, on a polished steel target where the matrix was laid in a line, and also in a spot. Refer to Section 9.6.1 for more detail on on each of these sample preparations.

Visually, the sonicated dried droplet and dried droplet preparations were very similar (Figure 6.1A-D). Those prepared on a polished steel target beaded during crystallisation, resulting in areas rich in matrix/sample and areas within the sample spot where no sample was present (Figure 6.1A & C). This irregular distribution makes these preparations unreliable for automated data acquisition on a mass spectrometer, due to the non-uniform distribution of matrix/sample on the target. The sonicated dried droplet and dried droplet preparations on an anchorchip showed a more uniform distribution of matrix/sample, as the droplet was held in place by the hydrophilic spot as it dried (Figure 6.1B & D). This would mean that matrix/sample would be present anywhere within this sample spot if automated data acquisition were undertaken.

146

Figure 6.1: Digital images of the sample preparations used in this study.

Images acquired using the charged-coupled device camera installed in the mass spectrometer.

The thin-layer preparation performed on an anchorchip was much smaller than the other preparations, as the matrix was restricted to the hydrophilic spot on the anchorchip (ca. 600 µm diameter; Figure 6.1E). The thin-layer-line preparation had small crystals of matrix on the surface of the polished steel target and a lighter circle where the sample had been laid and the matrix recrystallised (Figure 6.1F).

The line through the centre was where the auto-pipette tip was dragged down the length of the target during preparation of the thin layer of matrix. The

thin-layer-147

spot preparation looked similar to the thin-layer-line preparation, in that it had small crystals of matrix on the surface of the target and a lighter circle where the sample was laid, but it lacked the line down the centre (Figure 6.1G). This was because each spot was formed individually from a drop of matrix dissolved in acetone, making the technique suitable for automated sample preparation.

An assessment of the spot-to-spot reproducibility for each of the seven preparations was performed using the peak height ratio (PHR) of 2.5 µM [Dha⁷] MC-LR/50 µM angiotensin I. The coefficients of variation (CVs) reported in Table 6.1 are the average of three separate analyses performed using fresh preparations on different days. High levels of reproducibility were not achieved using both the sonicated dried droplet and dried droplet preparations on either the polished steel or anchorchip targets, giving CVs of around 20%. During the analysis of the thin-layer preparation on an anchorchip, it was noted that both microcystin and angiotensin I signals were low in the centre of the sample spot and increased in intensity towards the outside edge. This preparation yielded a CV of 22.5%. The two thin-layer preparations performed on a polished steel target gave good signal reproducibility, returning CVs of 11.9% for the line preparation and 9.5% for the spot preparation. The thicker line of matrix in the thin-layer-line preparation (Figure 6.1F) did not affect the signal compared with the rest of the sample spot.

Table 6.1: Performance of seven MALDI sample preparations.

Sample Preparation CV^a

(%)

MDL^b

(µM)

Sonicated dried droplet: Polished Steel 18.7 0.100 Sonicated dried droplet: Anchorchip 18.8 0.088

Dried droplet: Polished Steel 18.1 0.086

Dried droplet: Anchorchip 24.8 0.228

Thin-layer: Anchorchip 22.5 0.073

Thin-layer-line: Polished Steel 11.9 0.077

Thin-layer-spot: Polished Steel 9.5 0.087

Values are the average of triplicate analyses conducted over three days using fresh sample preparations. ^a CV = Coefficients of variation; determined using 2.5 µM [Dha⁷] MC-LR.

b MDL = Method detection limit for [Dha⁷] MC-LR; as per Section 9.6.3.

148

The method detection limit (MDL) was used to assess the sensitivity of each preparation (Table 6.1; Refer to Section 9.6.3 for more details on how the MDL was determined). The sonicated dried droplet preparations had detection limits of 0.1 µM [Dha⁷] MC-LR on polished steel and 0.088 µM on the anchorchip. The dried droplet preparation performed on polished steel displayed a similar sensitivity, with a detection limit of 0.086 µM, but only 0.228 µM when using the anchorchip. The thin-layer preparation performed on the anchorchip had a detection limit of 0.073 µM, but required a higher laser power than the other preparations, to ionise the microcystin/angiotensin I. The thin-layer preparations on polished steel performed well, with detection limits of 0.077 µM for the line preparation and 0.087 µM for the spot preparation.

The performance of the thin-layer-spot preparation was also assessed using two other microcystin congeners; MC-RR and MC-YR. Both MC-RR and MC-YR yielded good CVs on the thin-layer-spot preparation (6.2% and 6.8%, respectively;

Table 6.2). The MDLs were 0.031 µM for MC-RR and 0.056 µM for MC-YR.

Both the reproducibility and sensitivity for these two congeners were better than that observed with [Dha⁷] MC-LR (CV 9.5%, MDL 0.087 µM).

Table 6.2: Quantitative performance of the thin-layer-spot preparation performed on polished steel using three microcystin congeners.

Microcystin CV^a

a CV = Coefficients of variation; determined using 2.5 µM microcystin. ^b MDL = Method detection limit for each microcystin. ^c Quantitative ranges begin at the limit of quantitation.

The quantitative performance of these three microcystin congeners was also tested using the thin-layer-spot preparation. Standard curves for each of the individual microcystins were constructed using microcystin concentrations in the range of 0.1-10 µM. Each microcystin congener showed good linearity over this range, with R² values between 0.98 and 0.99 (Table 6.2).

149