ESDeposition

Moderator: Kevin Owens

This wiki is meant to be a resource for those looking for information on how to prepare samples for matrix-assisted laser desorption/ionization (MALDI) analysis by electrospray (ES) deposition. Our research group has found this means of sample preparation VERY important for producing homogeneous samples- samples where replicate mass spectra produce highly reproducible absolute peak areas (i.e., with very low coefficients of variation).

Figure 1: Schematic diagram of the manual electrospray deposition apparatus in use in our laboratory.

ESDiagram6-06.jpg

Figure 2: Photograph of the manual electrospray deposition apparatus.

ESApparatus1.jpg

Figure 3: Photograph of the Taylor cone formed on the end of the HPLC tubing used as the ES needle.

ESApparatus2.jpg

Electrospray Deposition Apparatus Details

HV power supply
Bertan PMT-75C-P-3 0, a 250 uA precision PMT power supply module (output: 0-7500V)

We’ve built this into a standalone HV supply including a digital readout (4-1/2 digit LCD display from Acculex), including a 10 turn potentiometer for voltage adjustment. Good filtering is a must- we use a three pole LC filter on the HV output. We added a HV relay to the HV output line (from Gigavac, Inc) activated by an operate/stand-by switch. This allows the HV supply to remain on for increased voltage stability. The electrospray apparatus is enclosed in a custom-built safety cabinet constructed of plexiglass. A microswitch interlock on the door is connected in series with the operate/stand-by switch for added safety.

Pumping System
Harvard Apparatus model 22 infusion pump (although any syringe pumping system with microliter/minute flow rates is suitable). The pump we use is unidirectional- a bidirectional system would have advantages for easier re-filling of the electrospray capillary.

Procedure
Mix sample at the usual concentration with matrix (we use approximately 0.10M) in the selected solvent. Add any ionization reagents (e.g., Na trifluoroacetate (TFA) for oxygen containing polymers such as polyethylene glycol), if necessary, but remember, as the sample segregation is decreased, you will need less ionization reagent than for a dried-droplet preparation. The mixture is sprayed directly onto the MS probe. We have sprayed methanol, ethanol, isopropanol, acetone, THF, and HFIP solutions with success.

Electrospray is accomplished by introducing approximately 2.5 µL of sample mixture into a small bore stainless steel capillary, 0.01 inches internal diameter, and approximately 100 mm in length (HPLC tubing, Upchurch Scientific). A potential of approximately +5 kV is applied to the capillary while the MS sample probe tip is held at ground a distance of 2.5 cm away. The MS probe is part of the grounded block- in our setup this is approximately 2.5” in diameter. A flow rate of approximately 2 µL per minute is applied using the syringe pump. Generally, between 1 and 2 µL of any given sample is electrosprayed- we have noted recently that thinner samples perform better than thicker samples. With this setup the circular spray pattern on the probe will be approximately 2-2.5 cm in diameter. If this is too large for your sample holder, we have found wax paper with a hole punched of the right size works well as a mask. The thickness increases from the edge to the center- our estimation of the center thickness is around 100A for a 30 second spray (measured by ellipsometry). If you observe diffraction rings on the sample spot (when viewed in ambient light) it is likely a good spray.

Note that the distance between the capillary tip and grounded probe, the flow rate and the applied voltage needs to be adjusted to obtain a stable, elongated Taylor cone. Round droplets at the end of the capillary indicate too low an applied potential; multiple small Taylor cones indicate too high a potential. The flow rate also is adjusted according to the solution conditions to avoid what we call sputtering (it is what it sounds like). As you switch from one solvent to another and one matrix (and matrix concentration) to another you need to adjust these variables to find where a good stable spray is obtained and where good MALDI results are observed (hey-this is called method development).

Our observation is that you should NOT see any crystals on the probe, either by eye or by moderate optical magnification. In fact, we have not seen crystals formed under good ES conditions either by SEM or TOF SIMS with a resolution of approximately 0.5 um. If you do see crystals, the droplets are too big as they hit the probe, and sample segregation will still be a problem.

References
Russell R. Hensel, Richard C. King, Kevin G. Owens*, “Electrospray Sample Preparation for Improved Quantitation in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry", Rapid Communications in Mass Spectrometry, 11(16), 1785-93, 1997.

Russell R. Hensel, Ph.D. Thesis, Drexel University, 1996.

Cynthia Chavez-Eng, Ph.D. Thesis, Drexel University, 2002.