Hi John,

for MALDI-TOF there can be various ion species occur, e.g. [M+H]+, [M+Na]+, [M+K]+ [M+NH4]+... Sometimes you can find all of them for the peptide, sometimes not. There is also the possibility to form species like [2M+H]+, [3M+2H]2+, [M+H+Na]2+... During my research I found out, that especially hydrophobic peptides do not ionize to the [M+H]+ ion, for those I can only find  [M+Na]+, [M+K]+, although i add TFA or other acids to the sample solution. Please notice the images attached (spectra took on a Bruker Autoflex speed TOF/TOF system, Matrix HCCA, solvent 30% AcN/Water + 0.1 % TFA). The first is a peptide which has an error in the sequence (one Glycine missing)  with all species [M+H]+, [M+Na]+, [M+K]+ and the other is hydrophobic peptide (isotopic mass of 1461,5)  where I only find [M+Na]+, [M+K]+ (mass difference between  [M+Na]+ and [M+K]+ = 16).

Another reason could be that the sample is simply not ionizable (is that a right english word?) in the matrix mixture you use. The sample simply does not "fly" to the detector. Try different matrices, solvents, acids, salt additions (like Na-TFA) and concentrations. Often, it is trial-and-error method.

Last reason which comes in my mind is that the ions you produce are not stable and fragment during their flight in the TOF mass analysator. If you have the opportunity, try linear mode instead of reflectance mode - you'll loose some isotopic resolution, but maybe gain more Ions.

Hi John,

for MALDI-TOF there can be various ion species occur, e.g. [M+H]+, [M+Na]+, [M+K]+ [M+NH4]+... Sometimes you can find all of them for the peptide, sometimes not. There is also the possibility to form species like [2M+H]+, [3M+2H]2+, [M+H+Na]2+... During my research I found out, that especially hydrophobic peptides do not ionize to the [M+H]+ ion, for those I can only find  [M+Na]+, [M+K]+, although i add TFA or other acids to the sample solution. Please notice the images attached (spectra took on a Bruker Autoflex speed TOF/TOF system, Matrix HCCA, solvent 30% AcN/Water + 0.1 % TFA). The first is a peptide which has an error in the sequence (one Glycine missing)  with all species [M+H]+, [M+Na]+, [M+K]+ and the other is hydrophobic peptide (isotopic mass of 1461,5)  where I only find [M+Na]+, [M+K]+ (mass difference between  [M+Na]+ and [M+K]+ = 16).

Another reason could be that the sample is simply not ionizable (is that a right english word?) in the matrix mixture you use. The sample simply does not "fly" to the detector. Try different matrices, solvents, acids, salt additions (like Na-TFA) and concentrations. Often, it is trial-and-error method.

Last reason which comes in my mind is that the ions you produce are not stable and fragment during their flight in the TOF mass analysator. If you have the opportunity, try linear mode instead of reflectance mode - you'll loose some isotopic resolution, but maybe gain more Ions.

-missed cleavage

glycan attached to peptide

other post-translational modification which alter the molecular weight

oxidized cys/methionine add mass of oxigen (this can be nicely demonstrated using a hair dryer so dry your sample)

Kind regards,

Jens

http://www.biomarker-discovery.com/

Hi Marcus,

One idea would be to add 0.1-1% n-octyl-glucopyranoside to your sample: as Chait already showed, the use of non-ionic surfactants won't effect the MS quality but will improve the detection of membrane proteins. Breaux showed also that OG will be very useful for analysis of hydrophobic peptides without surfactant adduct formation.

It is also important to be sure to keep your sample in solution prior to MS...

And I agree that playing with matrices and sample/matrix preparations is very important since each matrix has a different behavior in desoption/ionization.

Hope it helps :)

Stella 

I gather you are referring to missing peaks in the spectrum, based on theoretical digest of the protein sequence. 

You mentioned that it is a 'known' protein, but the spectrum is from an in-gel digest of a band. Variety of factors play a role in the final observed spectrum/spectra irrespective of the methods (maldi-tof or lc/ms) used. Let's limit the discussion to maldi-tofms method. 

Is the protein glycosylated or has other post-translational modifications? If modified, is the modification quantitative (for example, oxidation of methionine tends to be partial that complete)?

Does the band has a single protein?

Is the protein of full length or truncated compared to the theoretical sequence?

Is the reduction/alkylation and enzymatic digestion complete? When comparing to a theoretical list of expected peptides, is consideration given to artifacts of partial digestion at Lys-Pro and Arg-Pro motifs?

Did all the peptides from the digest get extracted into solution being analyzed? 

Once the digest sample is spotted along with the matrix, did all peptides get incorporated in the matrix crystals?

As mentioned by the others, the choice of matrix, presence of salts in the sample, ionization efficiency of peptide, mode (linear vs reflector) of analysis all play a role in the final spectrum observed. Sometimes a single pure peptide may give a good spectrum, but the signal may be suppressed when it is present in a digest. Choice of instrumental conditions like low mass gate can help you to 'see' a peptide of interest.

It is always useful to analyze/compare with spectrum obtained from a solution digest of the same pure protein. 

If the purpose of the experiment is to ID the gel band - protein truncations, partial modifications, incomplete digestions, in-efficient extractions, suppression of signals from some peptides - may not affect the outcome. Your result might say the major component in the gel band is related to protein X.

Here we are not even considering the instrumental capabilities of sensitivity, resolution and mass accuracy.

If the purpose of the experiment is to characterize the protein - get complete sequence coverage, understand the site-specific modifications, etc - then one has to do multiple experiments as suggested in the discussion thread.

Hope it helps :)

Viswanatham

Maybe your problem is solved already, but I found r really nice question regarding that topic, I add the link. Gerrit van der Honing (3rd answer) gave a really huge excel file where you can find a whole buch of contaminant ions or other species for your MS. This may help to explain something.

https://www.researchgate.net/post/What_may_be_the_reason_if_I_am_getting_higher_mass_calculated_mass_42060_experimentally_447_in_mass_spectrometry