When I analyze peptides in MALDI TOF sometimes I observe exactly the mass in the first peak of the isotopic envelope (not M+H). Is it correct? For example: my peptide has 2111.20 g/mol and M+H is 2112.21 ( pep-calc simulation). I observed in MALDI TOF that the first peak of isotopic envelope 2111.30 and the second 2112,3. Is this my peptide? The first peak should be 2112,3? Thank you so much for the help!
Dear Vanessa,
I suspect that you are confusing average mass with monoisotopic one. MALDI-TOF in reflectron mode resolves isotopic peaks of peptides thus you should not use average masses for mass spectra interpretation. In the mass range you use (at ~ 2 kDa) the difference between monoisotopic and average masses due to mass defects of elements is ~ 1 Da, this leads to the wrong identification of M+H as M+.
You should review mass definition as used in mass spectrometry. You wrote that your peptide has 2111.20 g/mol, which refers to molar mass or molecular weight and not to molecular mass. Molecular mass, in mass spectrometry, is usually referred as monoisotopic and/or average masses, defined in Dalton (Da) or unified atomic mass unit (u). In a mass spectrum, the correct notation for a monocharged positive ion is [M+H]+
For a good understanding of that please visit
https://www.ionsource.com/tutorial/isotopes/slide8.htm
With MALDI and suitable matrix, such as 4HCCA, your peptides will go to gas phase and then, with TOF reflectron resolution around 10000 FWHM, compound ions with isotopic distribution shall be detected. A good external calibration, with at least 5 calibrants covering the mass range you are interested in, is mandatory! As you perform calibration you should check your error/Da, which may be around 0.05 or less. If your error/Da is close to 1 you should discard your mass spectra, and check your calibrant quality and/or instrument settings.
Answering your question, if you have observed 2111.30 as the first peak of a group of ions with an aceptable and usual isotopic distribution (envelope) at 2000 Da - the second peak slightly higher in intensity than the first one - then 2111.30 shall be your experimentally observed monoisotopic mass. In this scenario the second peak (2112,30) can not be the first. However, if the intensities of your peaks are odd, you should consider the presence of two different peptides resolving at 2111 and 2112, with mass difference of 1 Dalton.
Considering that you have only one peptide/compound resolved at that m/z and if your theoretical monoisotopic mass (2112.21) is truly ~1 Da higher than the observed one (2111.30) then I think your experiment is not calibrated at all/calibration is poor at the 2000 Da mass range. Otherwise, if your calibration is good and everything is fine with your instrument then you should check the theoretical monoisotopic mass calculation.
You may also check your mass error and theoretical m/z at the following page:
https://warwick.ac.uk/fac/sci/chemistry/research/barrow/barrowgroup/calculators/mass_errors/
Good luck!
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