I am trying to detect very large proteins by MALDI MS (in linear mode) using superDHB. Even if I see the protein on the gel, I am struggling to get any useful spectra. I read that super DHB in combination with 5-methylsalicylic acid can be an option: does anyone have experience with that? I am going to try other matrices but it would be great if someone could help me to understand why I got such poor spectra.
Hi! I think it is very difficult to detect proteins of high MW using MALDI. However, in the past I could observe quite easily BSA (66 kDa) using a modified thin-layer method with aCHCA. Yep... with aCHCA. I used the method reported by
Cadene and B.T. Chait, A robust, detergent-friendly method for mass
spectrometric analysis of integral membrane proteins, Anal. Chem., 72 (2000)
It might help.. but not sure.
JLuis
Thank you Jose.
I know, the ionization of big masses is pretty unpaired; however, some good work was published by Zenobi et al and I believe there is a way....Roepstoff published few years ago the sDHB as a great alternative for intact proteins, especially because decrease the rate of fragmentation at mass>50kDa. I was wonder how the others manage to get that matrix working.....CHCA is an interesting point, due to the fact that is pretty acidic an "hard", which will induce high fragmentation,thus better for peptides than intact proteins..I was more thinking about DHB or DHAP...again SA is ok but the others are much nicely tolerant to salt...anyway, I am going to follow your suggestion as well.
Thanks again :)
Cheers
Stella
Hi Stella... Yes, it is a challenge to get nice spectra for high MW proteins. I tried the method of thin-layer when I was in graduate school at Michigan State University in the Jack Throck Watson group (you may know one of his books). It was a surprise to get a nice signal from BSA (according what I remember). However, our interest at that time was with peptides and proteins of low MW (less than 20 kDa), so I never tried other big proteins. At any case, I like the TL method because it seems there is a focused crystallization which provokes better MALDI spectra. About 2-3 years ago one of my former students tried to applied it to other matrices but He was no successful during the first attempts and just stopped to try :( . Good luck!
Best.
JLuis
How much (in pmol) amount of your proteins did you spot for MALDI MS? Very large proteins require more quantities than peptides or small proteins to obtain enough signals.
CovalX designs detectors with amazing high mass sensitivity. ABSciex in framingham/nadick had one on a ABSciex 5800 that could accurately detect 1,000,000Da. Their website has all the info. I recommend contacting them and asking who around you has their instrument adapted in this way OR if they would run a few samples for you as a courtesy to demo the instrument. I'd be surprised if they said no. I also have detected large proteins with CHCA but dried under a vacuum and recrystallized. (there is a 5800 next to me in my avatar pic).
All my best,
Chris
Try this site:
http://www.covalx.com/services
Sigma Aldrich has a great application note about matrices. Based on said note, I would try sinapic acid and 2-(4-hydroxyphenylazo)benzoic acid (HABA). There are other suggestions and references within that note, so it may be a good resource (it is attached to this comment).
First of all you have to calibrate the instrument Ig G protein. You must use sinapic acid as matrix to get good ionization and play with guide wire and grid voltage (should be increased). please try and reply.
Hi! How about using a "super-charging" reagents? I heard they are great for ESI MS. You may try to experiment them together with MALDI matrix.
Hi Rofeamor, that is a great idea: I tried DMSO but the limitation in ionization, make me to use very low %, which deals with no differences.....do you have any experience with those reagents in MALDI? What about the behaviour of mNBA?
Cheers
Stella
How was everything? Did you get some good results? By the way few months ago I observed a MALDI spectra of a protein about 120 kDa obtained with a MALDI TOF/TOF 5800 in the mass spec facility of UC Irvine. You can contact Dr. Benian Berhane for some information about the method used. http://www.chem.uci.edu/~jgreaves/mass-spectrometry/index.php/personnel. Best regards
Hi Maria, some good suggestions in here for choice of matrix (Sinapinic, CHCA, DHB, sDHB, HABA). You might want to add Ferulic Acid, which works similar to Sinapinic but sometimes gives better S/N for large proteins.
Not sure about the super-charging reagents - these are actually liquid and will keep your matrix from crystallizing. mNBA is an old FAB matrix, actually.
That is a good point, Christof! Thanks, I will try Ferulic acid next. Hope it works better than SA, which I am struggling with, especially if the sample contains salt....For now I am trying to get doubly-triply charged masses using DHAP, which is helping to get more accurate calibration, since resolution is much better at lower m/z.
Thanks a lot to all for the very useful comments and suggestions ;)
I was used the HCCA and SA as a matrix on MALDI target plate but unfortunately is difficult to identify intact proteins by MALDI-TOF MS due to their very large molecular weight. Is better to analyse the peptide mass mapping from the peptide analysis on the reflection mode of the MALDI TOF. Due to the high molecular weight of intact proteins low resolution peaks were generated. The best peak signals were generated in peptide mass mapping.
Hello Maria,
I can see this discussion started long before and i hope you have get better ion intensity for those high mass proteins. Could you please share your update regarding to your MS analysis of high mass protein. I have tried detecting BSA and IgG with SA matrix by double layer sample preparation method and I could see intensity of 5000 plus for BSA and 3000 for singly charge IgG . It is very important to maintain proper ratio of analyte to matrix in order to improve intensity.
Hi all,
Maria Stella Ritorto could you share your opinions on matrices for big proteins?
I am also working on a MALDI (UltraFleXtreme) with large proteins, quite chalenging. I can get peaks with sinapinic acid but it is noisy above 100 kDa and the resolution is weak.
Best,
Sébastien
Dear Sébastien Brûlé , I am working on High mass protein analysis. I use Bruker's MIcroflex MALDI TOF on liner mode of detection. Sinapic acid is good matrix for high mass protein analysis. solvent. we usually prepare matrix 2 mG /100uL (DD water : ACN +0.1-1% TFA). Moreover you have to check whats the ions source 1, 2 and lens voltage. the proper combination of these settings leads to good intensity and resolution. All the best
- Badges
- Science topic