Hello friends,
I am really really desparate right now!
I am working in negative mode in Mass spec.
I added formic acid+acetonitrile+water to my protein and I didn't receive any signal.
I added NH4OH and water, I didn't receive any.
I added NH4acetate and I didn't receive any signal (even one small one!!)
I need to see a good signal.
What is your recommendations?
Thank you!
Best,
Hi Maryam, assuming that the buffer system you are using has low ionic strenght (i.e. 20-50 mM), the buffers you described should work fine. I would check if there is a problem with your protein sample or the MS detector (i.e. you can try to use a standard protein like bovine serum albumin as reference sample). Good luck!
MS requires a fragmentation pattern and placing a charge on this fragment (m/z). Once you have performed the above check the MS and whether it is 'strong' enough to do this. Not every target may be torn apart and charged!
Hello Josef,
I used NH4OH 0.3% .
I calibrate my mass spec with NaI.
You mean that I should change it to a bovine solution?
What should be the concentration?
I really appreciate you for you time and answers.
Best,
Don't assume that your protein elutes from the column, particularly if it LC/MS.
Hi Maryam - For proteins I would recommend a buffer solvent system which works in a pH range between 5 -8 - thererfore I think the NH4/formate or NH4/acetate should be fine - try to keep the ionic strenght low (20-50 mM). I suggested bovine serum albumin (BSA) as a standard reference sample because it is very stable in solution and it is easy to obtain and if you dont have it in your lab certainly most biochemistry or clinical labs on your campus will have it and can easily give you a small amount for your experiment. I would start with testing your solvent and instrument setup with quite low sample BSA concentrations, i.e. 10 ug/ml - if you dont see a signal, you can increase the concentration to 100 ug/mL and 1000 ug/ml. If you still dont see anything dont increase the concentration any further, as this would likely harm your LC-column. If you can not see this standard protein than i guess the most likely reason is that there is a problem with your instrumental setup. Good luck!
Dear Josef and Bruce,
Thank you very much!
Josef, I'm gonna start the experiments and I hope to see signals.
Best,
Ionization in negative ion mode requires removing protons from the analyte. Adding acids (formate, acetate, or TFA) goes the wrong direction. You also need a volatile buffer system to support the protein in solution. Pyridinium actetate is an old standby that works reasonably well. They pyridine acts as a base to scarf up the acidic protons from the protein and both the pyridine and acetate are more volatile than ammonium and carbonate, which tend to form salt clusters. Pyr CH2COOH also butters in the pH range that is generally good for proteins. 5-7. If you're still having issues, try substituting phenolate for acetate. As with the above responses, keep the buffer concentration as low as possible for best results (generally below 20 mM). If you hit a solubility problem, you might try adding some fatty acids (C8-C14) or fatty alcohols) to the solution. These can aid solubility by masking hydrophobic groups, yet remain volatile in the MS. Use very low doses however (
Is there a reason for using negative mode for protein analysis? These are most often done in positive mode. You might also try starting with a small protein, e.g., cytochrome C or myoglobin, since these typically work well on LC-MS (although usually in positive mode). Also, in our lab, we rarely try to analyze a research protein of interest until we're sure the system is working for something we're familiar with.
I second John and Luke - if you managed to get it work, why not also try positive mode? The negatively charged molecules from the buffer should not effect the sensitivity, as these small masses generally are not in the scanning area of the detector (i.e.
Positive ion mode is always more sensitive than negative. Buffers are always a problem in MS, they suck up the charge and form clusters with the analyte, so if you can get away without them do it and always use the minimum possible. Hard ion buffers (e.g., Na, K, Cl, PO4, etc) are always worse than weak organic ions, but always choose organics with the highest vapor pressures (more volatile). MS is more art than science and there is lots of mythology to wade through with very little scientific basis behind it. You might try testing your buffer systems with the nucleotide dT6, which is an old negative ion mode standard. If this works, then your buffer system is OK, if not, then try again.
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