Supplementary Material to the Article Entitled Improving the Signal Intensity and Sensitivity
Supplementary Material for “Improving the Signal Intensity and Sensitivity of MALDI Mass Spectrometry by Using Nanoliter Spots Deposited by Induction-based Fluidics”
Tingting Tu1, Andrew D. Sauter Jr.2, Andrew D. Sauter III2, Michael L. Gross*,1
Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, MO 631301
Nanoliter, LLC, Henderson, NV 890742
Contents
The supplementary information includes figures containing data from experiments showing the relationship of MALDI spot volumes, spot diameters and mass spectral signals using ionic liquid matrix butylammonium α-cyano-4-hydroxycinnamate (Bu-CHCA). The figure for the quantitative measurement of signal enhancement by CHCA nanoliter spots with the same amount of analyte is also shown.
Results
We compared the sizes of 200, 100, 50, 20, 10 nL spots with that of a traditional 0.5 µL spot. We found that the diameters of the droplets are roughly proportional to the cube roots of the volumes, suggesting that the spotter is giving accurate depositions (Table S-1). We also wished to verify that the spotting technique gave results that depend only on the concentration of the analyte and not on the volume of the spotting solution when an ionic liquid matrix was used. Therefore, we measured the signals obtainable by MALDI of six spots made by depositing different volumes containing the same concentration (Figure S-2). The signal intensity for each spot was the average of six measurements, each consisting of 1000 laser shots. The results verify that signals obtained by MALDI of nanoliter spots are concentration dependent when using ILM.
Figure S-1 shows the data acquired from normal size or nanoliter spots containing the same amount of analyte for depositing solid matrices. The three solid lines show the dynamic range for absolute quantitation of bradykinin based on the molecular ion signal intensity. The slopes of these three lines are 55 (lower), 384 (middle) and 563 (upper). The dashed line, with a slope 5 times that of the normal-size spot line, represents the expected signal enhancement proportional to the concentration enhancement for 100 nL spots without any “additional signal enhancement”. The gap between dashed line and actual line for 100 nL spots shows the “additional signal enhancement”, which might be attributed to the “depth effect” (as discussed in the main text).
Sample Deposition Volumes / Deposition Methods / Spot diameters (mm) / Relative Average Signals / Relative standard deviation0.5 mL / Pipettor / 1.5 / 1.00 / 12%
200 nL / IBF spotter / 1.2 / 0.98 / 4%
100 nL / IBF spotter / 0.9 / 0.98 / 9%
50 nL / IBF spotter / 0.6 / 0.96 / 7%
20 nL / IBF spotter / 0.5 / 1.02 / 7%
10 nL / IBF spotter / 0.4 / 0.97 / 10%
Table S-1 The relative signal intensities from spots with different volumes but the same analyte concentration. All the spots had a final bradykinin concentration at 5 µM and ILM concentration as 0.5 M. Spot diameters were determined relative to the known inner diameter of the sample well on the MALDI plate.
Figure S-1 Comparison of peak intensities for normal-size spots deposited by an Eppendorf pipettor (blue diamond) and IBF nanoliter spots (100 nL, pink square; 50 nL, green triangles) containing the same bradykinin amounts for MALDI using CHCA as the matrix. The three solid lines show the dynamic range for absolute quantitation of bradykinin based on the molecular ion signal intensity. The slopes of these three lines are 55 (lower), 384 (middle) and 563 (upper). To show the expected slope, the dashed line is also shown with a slope 5 times of that of normal-size spot line (see supplemental text for details).