MASS SPECTROMETRY IN BIOLOGY AND MEDICINE Appendix The Meaning and Usage of the Terms Monoisotopic Mass, Average Mass, Mass Resolution, and Mass Accuracy for Measurements of Biomolecules s. A. Carr, A. L. Burlingame and M. A. Baldwin Much confusion surrounds the meaning of the terms monoisotopic mass, average mass, resolution and mass accuracy in mass spectrometry, and how they interrelate. The following brief primer is intended to clarify these terms and to illustrate the effect they have on the data obtained and its interpretation. We have highlighted the effects of these parameters on measurements of masses spanning the range of 1000 to 25,000 Da because of important differences that are observed. We refer the interested reader to a number ofexcellent articles that have dealt with aspects ofthese issues [1-6]. MONOISOTOPIC MASS VERSUS AVERAGE MASS Most elements have a variety of naturally occurring isotopes, each with a unique mass and natural abundance. The monoisotopic mass of an element refers specifically to the lightest stable isotope of that element. For example, there are two principle isotopes of carbon, l2C and l3C, with masses of 12.000000 and 13.003355 and natural abundances of98.9 and 1.1%, respectively; thus the value defined for the monoisotopic mass of carbon is 12.00000 on this mass scale. Similarly, there are two naturally occurring isotopes for nitrogen, l4N, with a mass of 14.003074 (monoisotopic mass) and a relative abundance of99.6% and l5N, with a mass of 15.000109 and a relative abundance of ca. 0.4%. The monoisotopic mass of a molecule is thus obtained by summing the monoisotopic masses (including the decimal component, referred to as the mass defect) of each element present. Of course the measured molecular species (a measurement made on a large, statistical ensemble of molecules) will consist not only of species having just the lightest isotopes ofthe elements present, but also some percent­ age of species having one or more atoms of one (or more) ofthe heavier isotopes. The contribution of these heavier isotope peaks in the molecular ion cluster depends on the abundance-weighted sum of each element present. The theoreti­ cal appearance of these isotope clusters may be precisely calculated by solving a polynomial expression [6], and there are now many commercially available programs that will do this automatically. On the other hand the chemical average mass of an element is simply the sum ofthe abundance-weighted masses of all of its stable isotopes (e. g., 98.9% l2C and 1.1% l3C, to give the isotope weighted average mass of 12.011 for carbon). The average mass of a molecule is then the sum of the chemical average masses ofthe elements present. The peak top mass is the mass of the peak maximum ofthe isotope cluster. The relationship 553 APPENDIX Resolullon = 25000 2529.913 Peak Top Mass = 2530.91 Average Mass =2531.67 Monoisotopic Mass 2524 2525 2526 2527 2526 2529 Resolullon = SOOO Peak Top Mass = 2530.91 Average Mass = 2531.67 2524 2525 2526 2527 Resolution = 1000 Peak Top Mass = 2530.93 Average Mass = 2531 .67 2524 2525 2526 2527 Resolution = 500 Peak Top Mass = 2531 .15 Average M... = 2531.67 2524 2525 2526 2527 Resolution = 250 Peak Top Mass = 2531.43 Average Mass = 2531 .67 2524 2525 2526 2527 2528 2529 2530 2531 2532 2533 2534 2535 2536 2537 2538 2539 Fig. 1. Oxidized b-chain, insulin. Formula C97Hl5lN25046S4' The molecular ion cluster for the oxidized b-chain of insulin is shown at various resolutions. The asymmetry of the cluster becomes less apparent as the resolution is decreased and the peak top mass and the average mass become almost identical. between the monoisotopic mass, average mass, and peak top mass (sometimes referred to as the maximum mass) is illustrated in Figures 1 and 2 for a peptide of mass ca. 2500 and a protein with a mass of ca. 25,000, respectively. One important consequence ofthe contribution ofthe heavier isotope peaks is that for peptides with masses greater than ca. 2000, the peak corresponding to the monoisotopic mass is no longer the most abundant in the isotopic cluster (Fig. 1). With increasing molecular weight, the peak top mass continues to shift upward relative to the monoisotopic mass. Above masses of ca. 8000, the monoisotopic mass has an insignificant contribution to the isotopic envelope (Fig. 2). Whether the monoisotopic mass or the average mass should be used when measuring and reporting molecular weights will depend on the mass of the substance and the resolving power of the mass spectrometer. RESOLUTION The ability to separate and mass measure signals for peptides, proteins, etc. of similar, but not identical molecular mass is affected by the resolving power 554 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE Resolution ~ 5000 Peak Top Mass = 25518.91 Average Mass ~ 25519.58 Resolution = 1000 Peak Top Mass =25579.32 Average Mass = 25519.58 Resolution =25000 Peak Top Mass = 25519.04 Average Mass =25519.58 Resolution = 500 Peak Top Mass ~ 25579.48 Average Mass = 25519.58 2.5545 25550 25555 25560 25565 25570 25575 25580 25585 25590 25595 25600 25605 25610 25615 Fig. 2. Protein HIV-p24. Formula Cl12rJI1802N316033SS13. The molecular ion cluster for the protein HIV-p24 is shown at various resolutions. The position ofthe monoisotopic mass is indicated by the arrow. of the mass analyzer. Because peaks in a mass spectrum have width and shape, it is necessary to evaluate the extent of overlap between adjacent peaks when determin~gthe resolution. Mass resolution is often expressed as the ratio mILlm where m and m + Llm are the masses (in atomic mass units or Da) oftwo adjacent peaks of approximately equal intensity in the mass spectrum (Fig. 3). The height ofthe "valley" between the two adjacent peaks, expressed as a percentage of the height of peak "m", is a measure of the degree of overlap. There are presently two definitions in widespread use, and it is essential to know which is being used when resolution figures are quoted. Historically, the first of these is the so-called 10% valley definition in which the two adjacent peaks each contribute 5% to the valley between them (Fig. 3). In practice, it is often necessary to determine the resolution of the mass analyzer using a single peak (either one peak in a mass­ resolved isotopic peak cluster, or one peak corresponding to the envelope of unresolved isotopic peaks, see below). This is accomplished by measuring the peak width (in Da) at the 5% level, and dividing this number into the mass (in Da) of the peak. This definition is most frequently used for molecules with masses below 5000 Da where it is possible on certain analyzers to obtain 555 ApPENDIX h 8 M @50% h (FWHM) (10% Valley) M M+l Fig. 3. resolution of the isotope peaks. For unresolved isotopic peak envelopes the "full­ width, half-maximum" (FWHM) definition has come into popular use. The resolution of a peak using this definition is the mass ofthe peak (in Da) divided by the width (in Da) ofthe isotopic envelope measured at the half-height ofthe peak (see Fig. 3). A useful rule of thumb is that the value for the resolution determined using the FWHM definition is approximately twice that obtained using the 10% valley definition (equivalent for singly charged ions to the full­ width at 5% peak height for symmetrical peaks). For example, a resolution of 1000 using the 10% valley definition is approximately equivalent to resolution of 2000 using the FWHM definition: clearly, while the resolution value in the latter case is larger, the measured degree of separation of the peaks in Da is identical. It should also be noted that at a resolution of 1000 (FWHM) a peak at mass 1001 will essentially be unresolved from the peak at mass 1000. However, for proteins, resolving the isotopes in the protonated molecu­ lar ion envelope is generally not possible (except for FTMS of electrospray­ produced molecular ions), nor is it generally useful in practice to be able to resolve the isotopes oflarge molecules like proteins. Furthermore, increasing the resolving power of a mass spectrometer usually reduces the sensitivity of 556 MASS SPECTROMETRY IN BIOLOGY AND MEDICINE detection. Thus, in sample limited situations, the resolving power of the mass spectrometer may have to be reduced below the level necessary for resolution of the isotopic peaks to allow detection of the protonated molecular ion. MAss ACCURACY The accuracy of the mass measurement is often stated as a percentage of the measured mass (e. g., molecular mass = 1000 +/- 0.01%), or as parts-per­ million (e. g., molecular mass =1000 +/- 100 ppm). As the mass being considered increases, the absolute mass error corresponding to the percent or ppm error will also increase proportionally (e. g., 0.01% or 100 ppm = 0.1 Da at m/z 1000, or 0.5 Da at m/z 5000, or 5 Da at m/z 50,000). Mass accuracy is dramatically affected by how the data is treated by the data system. Very significant error can be introduced in the mass range of 1000 to ca. 5000 if unresolved isotope clusters must be measured. In this mass range, the unresolved clusters will be asymmet­ ric in shape, reflecting the asymmetry of the intensity distribution of the underlying isotopic masses in the cluster (see Fig. 1). Ifthe mass being reported is the average mass, one must be sure to measure the centroid of the distribution and not the peak top mass, as these will not generally be the same in this mass range. Errors in measuring the centroid can occur when the ion intensities are weak (due to the inaccurate definition of the peak profile due to poor ion statistics) or due to other distortions of the peak profile of the cluster.