We are using MWCO to fractionate our cell samples, then we evaporate our samples (to concentrate them) prior to digesting with trypsin. We have typically used N2 to dry the samples (inert atomosphere) but I'm looking for other methods. For example does anyone uses a speedvac to vacuum dry their samples for MALDI or LC/MS and what temperature do you use (e.g. do you heat your speedvac?) I'm not sure if I need to be worried about degradation at 40 degrees C (The typical max temp for speed vacs). Thanks!
There are many choices. You might try spin concentrators. Pierce sells a decent product. (http://www.piercenet.com/product/pierce-concentrators-pes-3k-10k-30k-100k-mwco)
If you want to take your sample to dryness, you could also lyophilize the samples.
We make extensive use of our SpeedVac, mostly for evaporating desalted peptide solutions after solid-phase extraction. We always evaporate at room temperature, that is, with the heater switched off. The big advantage of centrifugal evaporation over drying in a gas stream is that the dried material should be concentrated to the bottom of the tube, making re-suspension in small volumes of buffer easier.
Plus one for speed-vac, without heating. I do not heat the samples as I do worry about degradation. This should be less of a concern if you are dealing with peptides as opposed to proteins.
Hi Marianne,
I use speedvac at RT. If your samples are in small volumes like microliters (I assume so), I would first place the tubes in Dry ice freeze cold, and the speedvac, This shortens the time tor dry. Minutes will be enough to dry. While resuspending the sample, pipette many times up and down on the eppitubes walls.
We Lyophilize as well. Spin dry in a vacuum centrifuge with a cold trap attached ...
I have always used SpeedVac for drying the gel pieces after destaining with ABC buffer and ACN. Then treatment with Trypsin for 4 hours at RT and diffusion in ABC buffer overnight. Samples had been processed this way for both MALDI and LC-MS/MS. The final step before analyzing is different though.
Hi,
For bottom-up proteomics, you can use FASP for tryptic digestion. It combines sample concentration on a filter (10K or 30K, flat membrane required) and tryptic digestion. I have usually better results using FASP than in-solution digestion.
In a speed vac the samples are cooled by the evaporation of the solvent. Water usually freezes in the speed vac. However, when the sample is nearly dry, it heats up. If you do trypsin digestion, I would recommend not to dry the sample completely and just to concentrate them. The proteins can attach to the walls of your tube.
WOW, thank you so much for the comments, ideas and details! It is really heartening to know that many people are using speed vacs. Here are some answers to questions about my samples as well as a few more questions! (if you will permit me!)
Answer: We do drying both before and after digestion (our process right now is to separate our sample using molecular weight cut-offs, dry those "separated" samples, then perform a digest, then dry samples again until ready to spot.
Question A: "Lyophilization" to me, requires a dedicated freeze dryer, I am curious whether people are actually using a freeze dryer, and/or simply freezing their samples prior to running them in a speed vac at room temperature?
Question B: We do our enzyme digests at 37C for either 3 hr or overnight, so what is the harm in running the speed vac at 40C for 2 hrs to dry the samples? (Or at least, set it for 40C for the first hour while there is still liquid in the sample) [I'm knew to proteomics so please be kind!]
Thank you again for all the help. It is greatly appreciated!
Hi,
we are using a speedvac at 40 or 45 °C to concentrate the samples after digestion. In-solution digestions are usually desalted/purified by C18 STAGE beforehand. I see no problem to use a speed vac. There might be peptide loss if you dry your samples completely, but it's hard to avoid since some samples will dry faster than others.
I usually resuspend the peptides in 20 µL 0.1% formic acid and use a ultrasonication bath to get the peptides in solution. But take care, where the swimmer is touching the tubes it's getting hot. Long ultrasonication times might melt a hole in the tubes. I usually do it several times for 30 s.
It's also a good advice to use protein lobind tubes (high purity polypropylene). You will have less binding of peptides to the tube.
Regarding the drying of proteins: for some samples, it's hard to get dried or precipitated proteins back into solution, so they are accessible for digestion. From my experience, I would rather suggest concentrating than drying or precipitating. As already said, FASP works nicely. Most people work with 30K filters but you might loose some proteins. The Vivacon 500 or Microcon 500 filters are good for this purpose:
http://www.sciencedirect.com/science/article/pii/S000326971000775X#
After digestion, the peptides are eluted through the filter.
Check your speed vac whether it is refrigerated? So you can run the samples at 4 deg
Always use the refrigerated speed vac for lyophilized your sample.It is very important in case of protein samples.
i am trying to redissolve human saliva protein pellet for one and two dimensional electrophoresis. how should i resuspend the pellet i got from speed vac. should i use milli q water, phosphate buffer or 1 x running buffer to redissolve the pellet. pl suggest. thanks
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