Your mass calibration is off?

How did you calculate the theoretical mass?

Does the sequence contain asparagine residues that can deamidate? Or perhaps cysteines that form disulfide bonds?

Dear Jamila Ahmed,

You may want to review the data presented below:

Monoisotopic Mass

In fact, the monoisotopic mass is calculated by a peak-detection algorithm (SNAP 2.0, Bruker Daltonics), which fits an in silico isotopic distribution model, based on the average amino acid ‘averagine’, to the experimental data.

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/monoisotopic-mass

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Which one is the monoisotopic peak (e.g. peptides in MALDI-TOF)?

Question

• Asked October 27, 2014

According to the definition by Unimod

• "Monoisotopic mass is calculated using the mass of the most abundant natural isotope of each constituent element" and
• "Some sources suggest that monoisotopic mass is calculated from the lightest natural isotope of each element, but this is wrong. "

Attached is a figure (copied from a reference). For Panel a, it is quite obvious that the monoisotopic peak is the leftmost one.

So for Panel b, is the monoisotopic peak the one indicated by the blue or the red arrow?

Thanks.

http://www.unimod.org/masses.html

https://www.researchgate.net/post/Which_one_is_the_monoisotopic_peak_eg_peptides_in_MALDI-TOF

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What is the difference between average and monoisotopic mass calculations of peptides?

I often have to calculate the mass of peptide sequences using calculators such as here.

I have the option of calculating the average and monoisotopic mass, but what is the difference?

Answer:

The elements that typically make up peptides and proteins can be present in different isotopes. For example, carbon is present to 98.9% as 12CX12X2212C, and to 1.1% as 13CX13X2213C.

The monoisotopic mass assumes that every element is present in the most abundant isotope, the average mass uses the actual distribution of the isotopes in nature.

If you have sufficient resolution in your mass spectrum you can see multiple signals corresponding to different isotope compositions of your molecule.

https://biology.stackexchange.com/questions/5697/what-is-the-difference-between-average-and-monoisotopic-mass-calculations-of-pep

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Peptides are relatively large molecules, from the perspective of a medicinal chemist where anything larger than ~600 Da is "large". My experience is as one gets to 1000 Da and larger, the mass of a molecule starts to approach the average molecular weight of that molecule as each molecule is large enough to contain the average isotopic distribution of the common elements. The monoisotopic mass is calculated from the most common element, and large molecules are likely to contain a range of isotopes.

Hi Jamila,

A couple of practical points:

1. To compare calculated, monoisotopic mass with your observed, experimental mass, you have to be resolving your compound into its isotopes. Are you?

If not, you need to work on your laser power attenuation and method to do so. If it is difficult to resolve the sample into its C12, C13 etc. monoisotope peaks, you may want to try the linear mode on your MALDI-TOF MS to obtain average mass for your sample. Then, calculate the theoretical average mass for the peptide and see how close you are with that approach. Remember also that in MALDI-TOF MS, the m/z or experimental mass is always with a proton. So, all the calculated masses should have +H for comparative purposed.

2. If you have an experimental result that is looking heteregenous, you can try and smoothen the peak and the center of the peak is then your average, experimental mass, m/z

3. The last but most important point is your external calibration of the instrument. You need to use calibrants that cover your peptide mass and make sure that after you calibrate the instrument, save the calibration to your method (rename the method), acquire a new spectrum and check to make sure your calibrants are right on the theoretical masses. If not, recalibrate until you are happy with your calibration file.

Good luck!

Hediye.

Dear Jamila Ahmed,

I would rather suggest you go for deconvolution of the actual ms scan data and use pure substance. Use internal calibrator during data acquisition and that should be in your analyte mass range. For the sake of peptide mass calculations, the best resource is Protpi website