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Section 2B. ESI-MS Data

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    81227
  • One of the advantages of ESI is that it is a “soft” ionization technique in which little fragmentation of large, thermally fragile biomolecules occurs. Consequently, molecular weight information is readily obtained with this technique. Additionally, the ions formed are often multiply charged, which enables the analysis of molecular masses exceeding 100,000 Da because the multiple charges bring the m/z values into the mass range of conventional mass analyzers, such as ion traps. With proteins and peptides, the mass spectrum consists of a series of peaks, call the “peak envelope” which represents a distribution of multiply charged analyte ions.

    Ubiquitin is a small protein with a monoisotopic molecular weight of 8560 Da. Electrospray ionization of this small protein typically results in major charge states of +8, +9, +10, +11, +12, and +13.

    Reading Questions

    1. Using this information, complete the table below, assuming that the charges on each ion come from protonation rather than sodium or potassium adducts. Round masses and m/z values to the ones place.

    z mass of [M+zH]+z m/z
    8
    9
    10
    11
    12
    13

    2. Using the data you entered in the table above, sketch an expected ESI-MS spectrum for ubiquitin. Label each peak with its charge state. What do you notice about the spacing of the peaks along the x-axis?

    Based on the m/z values and peak spacing observed in the charge envelope, we can determine the charge state, z, for each peak and the molecular weight of the analyte using Equations \(\ref{1}\) and \(\ref{2}\),

    \[z = \dfrac{M_2-A}{M_1-M_2} \label{1}\]

    \[MW= \dfrac{(M_1-A)(M_2-A)}{M_1-M_2} \label{2}\]

    where MW is the molecular weight of the analyte, M1 is the m/z value for the first ion, z is the charge state of the first ion, M2 is the m/z value for a second ion of lower m/z, and A is the mass of the adduct ion, which is usually a proton (H+) but can be sodium (Na+) or potassium (K+) ions from glassware or buffers used in the experiment.

    Discussion Questions

    1. Why do aqueous samples for electrospray typically include an acid and a low surface tension solvent such as methanol?

    2. Figure 2 shows an experimentally obtained mass spectrum for ubiquitin. Compare this spectrum to the spectrum you predicted in Reading Question 2. Are there any differences? If so, what might cause these differences?

    Figure 2. ESI-MS spectrum of bovine ubiquitin from Protea Biosciences (proteabio.com/products/PS-143)

    3. Using Equation (\(\ref{2}\)) and any pair of peaks from Figure 2, calculate the molecular weight of ubiquitin and its percent error compared to the theoretical monoisotopic mass of 8560 amu.

    Figure 3. ESI-MS spectrum of cytochrome C. Data obtained at Trinity College.

    4. Figure 3 shows the ESI-MS spectrum for cytochrome C electrosprayed from a mixture of water, methanol, and acetic acid with pH of 2.5. What is the charge state of the peak at m/z = 773.36?

    5. Determine the MW of the analyte, cytochrome C, using the data in Figure 3.

    6. How would you expect the mass spectrum to change if the cytochrome C sample was electrosprayed from a solution of higher pH? Explain your answer.

    7. Note that in the ESI-MS spectra for ubiquitin and cytochrome C, each major peak is accompanied by a series of less intense peaks of slightly different m/z. What is the source of these peaks?

    8. Compare your description of the ion trap mass analyzer video with your group mates’ descriptions. As a group, write a consensus explanation for how an ion trap works.

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