Interpreting Electrospray Mass Spectra
"an IonSource.Com tutorial"
page 7

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Tips for Looking at Electrospray Spectra
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Frequently Asked Questions
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"Peak Tips"
 

"What I normally look for in a spectrum"

1) Look for the tallest peak in the spectrum above 200 mass units. (The low end of a spectrum can often be confounded with solvent noise.)  Then look for peaks that are roughly double or half the mass. This may tell you whether there are multiply charged species present which can help with mass determination.

  

2) Counts.  Resist interpreting meager spectra.  If you know that on a particular day that 1.0X106 is a respectable, reliable signal then you will know that a spectrum that tops out at 1.0X104 counts (or at background) may not be a reliable spectrum to interpret.  

 

3) The quality of the spectrum is important. You will waste valuable time interpreting a low quality spectrum.  If you are not happy with the quality of the spectrum try averaging several or many low level spectra to obtain a better quality "averaged mass spectrum."  Compare this to a background spectrum to see if the peaks really stand out.

 

4) Reproducibility is important.  The peak must be consistent to be considered a relevant peak. You should not lend credence to what I call "one scan wonders."

 

5) Determining the charge state of a peak when only one peak is obvious
A molecule will often have adduct ions associated with it other than hydrogen .  Look for sodium or ammonium adducts.  These adducts can often give you a hint as to the charge state of a peak.  For example if there is only one major species in a spectrum look for the sodium adduct following that peak.  If it is a singly charged species the sodium adduct will be found at +22 mass units higher than the M+H peak.  If the peak is doubly charged the adduct will appear at + 11 mass units.

Isotopes
If the mass spectrometer you are working with has sufficient resolution look at the isotopes, a singly charged ion will show isotopic peaks that differ by 1 mass unit, a doubly charged ion will show peaks that differ by 0.5 mass units and so on. This is another way to deduce the charge state of a peak and thus the mass. 

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Frequently Asked Questions

Q  How can I tell if a peak is real?
A Wow, this is some question.  All peaks are real.  In an LC/MS run we look for peaks that reoccur in multiple adjacent scans (spectrums) but not in every scan.  If the peak occurs in every scan it may be a background peak. It is possible to get system noise or spikes that only occur in one scan or sporadically these are most likely electronic or some other form of system noise. 
Q  How can I be sure of the identity of a peak.
A Well, a mass is just a mass and many compounds have isobaric mass so you can't be sure from just a mass.  In the old days we would perform an enzymatic digest on a protein, run an LC/MS peptide map and match up the mass with the theoretical fragments. Today the bar is rightly higher and we go one step further in the identification, we take the peak through a fragmentation and match up the fragment masses with the theoretical CID fragment masses for that peptide. This gives us a positive ID.

Another overlooked component in LC/MS is part of what makes LC/MS so powerful and that is the correlation of mass and LC retention time.  If the retention time of a molecule has been previously characterized this information can be linked with the mass information for a positive ID.

If you are characterizing a new molecule try modifying the molecule to see if you can modify the mass.  Try an enzyme digest if the unknown is a protein or try chemical modification if it is a small molecule and see if the mass of the unknown changes as predicted by the mass ID.

Q How can I differentiate a compound at one mass from another at twice the mass?  For example a compound with mass 1000 will display peaks at m/z 1001 and 501, and a compound with mass 2000 may display peaks at m/z 2001, 1001, 666.7 and 501.  The mass determination can further be confounded if the peptide at 1000 forms dimers during the electrospray process.
A 1) The peak envelope does not skip peaks, for example the 2000 mass even if it does not have an obvious peak at 2001 it should have the 666.7 peak between the 1001 and 501 peaks.

2) Also try to determine the charge state of the ions from the adducts or from the isotopes.  This will tell you what the mass of the compound is.

3) Dimer formation can be a major problem in some analyses. Try to reduce the concentration of the analyte. Often if the concentration is too high dimers will be observed in the spectrum.  Also dimers can be reduced by changing some of the setting on the mass spectrometer.

4) With the peak envelope of larger molecules (10kDa+) look for smooth peak distributions.  The peak distribution should have a smooth bell shaped curve appearance, sometimes trailing off to the right. The peak to peak relationship should be predictable, if one observes an alternating pattern of peak intensities this may be a clue to a coeluting dimer. 

Q I believe that I am observing artifctual fragmentation and do not know which peaks are real.
A Again all peaks are real. Sometimes on can induce fragmentation before the first mass filter (in-source CID, source CID). While this effect can be a valuable characterization strategy for those possessing single quadrupole instruments it is often a distraction when fragmentation is not the goal.  My suggestion is to try and slow the ions down so that they will not collide and fragment with the residual atmosphere in the source.  

                                    
 

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Last updated: Monday, July 18, 2005 01:43:20 PM