Wednesday, October 16, 2013

Do you need nanospray to do good proteomics? (Part 2)

I threw out this idea a while back, and some recent experiencies really make me want to reapproach it.

This week I worked with a core facility in Pittsburgh (which is quietly becoming a mass spectrometry powerhouse of a city, btw!).  At this facility, the majority of the work coming in is small molecule work, though nucleotides, whole proteins and shotgun work does make appearances.  Due to the chemical nature of the work, the HPLC in use is a high flow Ultimate 3000, and not enough shotgun proteomics is coming (yet) to justify the purchase of a nanoflow LC.

No problem.  Using the Peptide Retention Time Calibration mixture as a standard, we first benchmarked the sensitivity of the system (a Q Exactive with a 15 cm x 2.1 C-18 column, HESI source and 200 uL/min flow rate).  

I know this is hard to see, but this is 1 picomol of each of the 15 PRTC peptides.  The TIC baseline is almost 1E8.  At 1 picomol.  All 15 peptides came off nicely, when they should have.  The chromatography, obviously, could be improved, but our goal was to benchmark our sensitivity.

Next came the real sample.  A group down the hall digested a mouse liver (in solution), desalted and brought over a vial.  We set up a quick, first pass run to go overnight and came in this morning to this beautiful TIC.

Again, I apologize for the grainy JPEG.  The basepeak signal intensity is around 8E8.  These are quick runs.  Little time was spent optimizing the source conditions, dynamic exclusion, fill times, etc., we just injected a few different size aliquots of this digest.  The digest was 200 ug of mouse liver protein digested in solution and desalted.  If 0% loss, the above injection is 10 ug of protein.  Considering the losses involved due to membrane proteins (liver has an awful lot of membranes in it and no detergents were used) and to desalting, I would be surprised if we wee looking at 5ug of protein.

Yes, 10 ug on a nanocolumn is a lot.  But, come on!  10ug of protein is nothing for most biologists. You get 1-2 mg of protein from a T-75 flask of poorly growing adherent cells without even trying.

How are the results?  Using default percolator outputs provided, ~5550 peptides  and ~1250 unique protein groups.  You can't tell me that isn't awesome for a first pass, non-optimized 80 minute run.  You can, I guess, but I won't believe you.

Next, we took that run and exported a Q Exactive exclusion list through PD and put that in for an otherwise identical rerun.  Summing the two run resulted in ~1650 unique proteins and ~7000 unique peptides.  Not too shabby, in my opinion!

This experience was reinforced when I spent some time working with a big company recently.   Although I saw dozens of mass spectrometers doing proteomics experiments, I never once saw a nanoflow system.  I think that experiments of this kind are becoming more of a regularity than a novelty.  And the work this week demonstrates why.  Mass spectrometers are sensitive enough to work without the added sensitivity from nanoflow sources.  Unless your really need to be digging into the noise to look for the lowest copy number peptides and PTMS, electrospray or microspray may be enough to get you the the proteins that you need.  Nanospray will always result in higher signal, but sometimes you have to take a step back and think about just how much signal you really need.

BTW, these screenshots were graciously given to me by Dr. Bhaskar Godugu, Director of the Mass Spectrometry Facility (Chemistry) at the University of Pittsburgh.  

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