Electrospray Samples for MALDI
The SRS Mass Spectrometry laboratory is developing alternative sample preparation procedures to produce more robust and reliable sample spots for our potential customers. Currently, we are making matrix-assisted laser desorption ionization (MALDI) sample spots by electrospraying (ES) the analyte and matrix solution onto the sample plate. The volume of the spots ranges from 30 to 250 nanoliters (nL). When dry, the sample spots are less than 1 mm in diameter. Below is an image of the ES sample preparation process. The needle is approximately 0.75 mm in diameter. The characteristic “Taylor cone” of the solution is clearly visible at the end of the needle as well as the sample spot as it is drying.
There are numerous advantages of the ES spots over the conventional dried-droplet spots. First, the volume of solution used is an order of magnitude less than that which can be deposited by hand using a micro-pipette (approx. 1 microliter (uL)). The use of lower volumes allows for less dilution of the sample. This is significant because mass spectrometry is concentration dependent; that is, higher concentration typically results in better signal. Second, the analyte(s) is evenly distributed throughout the spot versus the conventional dried droplet sample preparation where the analyte partitions into various regions of the spot as the solvent evaporates. With the dried droplet sample preparation there are local “hot spots” requiring scanning of the laser beam around the sample spot. Below is an image of a dried droplet sample spot from 1uL of solution. The droplet is larger than 2 mm in diameter and contains many regions of localized matrix and analyte crystals.
The advantage of increased sample homogeneity of the ES spot is that wherever you irradiate the spot with the laser beam, analyte ion signal is generated; therefore, scanning the surface for analyte ion signal is not required. Below is an image of an ES sample spot from 100 nL of solution. The diameter of the spot is less than 1mm and there are no localized regions of matrix and analyte crystals. To see more about the homogeneity of the ES sample spot see MS Imaging of ES Sample Spot.
When using a dried-droplet sample preparation, the partitioning of the analyte molecules into different regions of the spot does not permit equal sampling of all components. However, using ES to make the sample spots, equal sampling of all components is achieved. The best example of this is observed when analyzing peptides produced from the enzymatic cleavage of a protein. Using the dried-droplet procedure, only a fraction of the peptides are detected in any given acquisition. The result of this fractionation is an incomplete coverage of the amino acid sequence of the protein (30-50%). When the ES sample preparation is used, most, if not all, of the peptides are detected and the coverage of the amino acid sequence of the protein is better than 90%.
Signal reproducibility from laser-pulse to laser-pulse is also very good with the ES sample preparation. Below is a plot of 10 single shot spectra of the isotopic distribution of a peptide ion from a bovine serum albumin (BSA) digest mixture. Notice the consistency in intensity of the ion signal for all the shots. Future experiments will be aimed at characterizing the pulse-to-pulse reproducibility at a various positions on the same sample spot as well as on multiple sample spots. These results will be compared to those obtained with the dried droplet sample preparation and a complete statistical analysis will be performed to substantiate the role of the ES sample preparation in MALDI.
A description of this work was presented at the June 2000 conference.