There are two samples MBP1 and MBP2 which are same samples of high concentration of Maltose Binding Protein (3-4 mg/ml). The other flatter samples is basically same sample but with 1 mM Hydrogen TetraChloroAurate (for source of Au^+3 ions).
Each sample contains follwoing salts:
Tris : 600 µM
NaCl: 750 µM
gold : 1mM (only in flatter profile)
Maltose: 150 µM
I dont know why even at this high concentration of protein and low salt I still do not get sharp peaks in MALDI profile.
CHCA was used as medium.
I am not that surprised of the spectra. You have a fairly large protein, and protein peaks will often broaden with increasing mass. What you have is probably a mixture of a lot of different adducts and protonation states (not charge states). Since it is furthermore a maltose binding protein (how many units can it bind?), that will also contribute to the mass distribution. If you use the centroid mass, you will get the average state of the protein. The above becomes even worse, when you chelate gold, as it will not bind in a defined manner but rather make a distribution of bound gold atoms. What is the resolution of the MALDI? I would expect to see an idication of a gold distribution, but since you have that many salts in there, the adduct distribution will be all over the place. With this data, the best you could do is to use centroid mass to determine the average number of bound gold. You could also use FWHM to get an estimate of the predominant range for gold distribution. Are the salts and maltose needed? If omitted, you should be able to get much cleaner and resolved spectra.
I am not that surprised of the spectra. You have a fairly large protein, and protein peaks will often broaden with increasing mass. What you have is probably a mixture of a lot of different adducts and protonation states (not charge states). Since it is furthermore a maltose binding protein (how many units can it bind?), that will also contribute to the mass distribution. If you use the centroid mass, you will get the average state of the protein. The above becomes even worse, when you chelate gold, as it will not bind in a defined manner but rather make a distribution of bound gold atoms. What is the resolution of the MALDI? I would expect to see an idication of a gold distribution, but since you have that many salts in there, the adduct distribution will be all over the place. With this data, the best you could do is to use centroid mass to determine the average number of bound gold. You could also use FWHM to get an estimate of the predominant range for gold distribution. Are the salts and maltose needed? If omitted, you should be able to get much cleaner and resolved spectra.
I would say that resolution of your spectra is good, as expected for protein analysis by MALDI-TOF linear mode at such a mass range.
Have a look to this article:
Article Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O...
To improve spectra resolution you may try the following:
- desalt your protein -> you may use a centrifugal filter - you will probably lose protein but MALDI-TOF is very sensitive;
- desalting or not, you should try a better preparation of protein/matrix layer on target -> you may test other matrices suitable for intact proteins and glycoproteins, such as sinapinic acid (SA) in double layer procedure - prepare a very thin matrix layer and apply sample so that the sample is on the outer layer of matrix - this helps to remove salts either;
- test 2,5-dihydroxy benzoic acid (DHB) or 2,5-dihydroxy acetophenone (DHAP) matrices;
- try AnchorChip targets -> specially if using DHB;
- adjust laser power and use Pulsed Ion Extraction, if available on your instrument.
I have no experience with gold particles but maybe you should have a look to these:
Article A new matrix of MALDI-TOF MS for the analysis of thiolate-pr...
http://orig04.deviantart.net/5644/f/2015/166/b/9/why_i_ve_been_so_busy____by_schwartze-d6yrtpb.pdf
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