Since version 2.2, MS Search (NIST) software can process mass spectra containing floating-point m/z values. Earlier versions of the program used to work only with nominal mass spectra; as a result, floating-point m/z values had to be converted to integers during import. Nowadays, a user can choose which behavior is preferable (Fig. 1) by selecting one of the options: "EI (nominal m/z)" or "Accurate m/z".
Fig. 1. The "Spectrum Import Options" window (to open this window select "Spectrum Import Options" item from the "Options" menu).
The "EI (nominal m/z)" option is used when converting to nominal mass spectra is required. Unfortunately, a detailed description of the conversion procedure is not provided in the MS Search User's Guide [1]. For example, it is not clear what happens when several floating-point m/z values are rounded to the same integer during import. In the case of GC/MS data, it is a common practice to sum all intensities of such peaks. However, we found out that MS Search 2.4 keeps only the most abundant peak and omits all the others (such behavior is typical in the case of LC/MS data). Our considerations can be illustrated using model mass spectra (Fig. 2). The import_ex1.msp file allows anyone to check our conclusions. The original mass spectra contain several peaks (with m/z 60.1, 60.2, and 60.3) which all are rounded to m/z = 60 (when default options are used). Comparing intensities of mass spectral peaks shows that some isobaric peaks are discarded during the import process.
Fig. 2. Importing mass spectra using the "EI (nominal m/z)" option.
The Accurate m/z option allows importing floating-point m/z values without rounding (normalized intensities are not rounded to integers either). NIST’20 database contains only nominal EI mass spectra. For this reason, experimental mass spectra containing floating-point m/z values have to be converted to nominal ones before library searching. Just like in the previous case, the MS Search User’s Guide [1] does not shed light on the back end of this conversion. We assumed the rounding algorithm to be the same as in the previous case. However, some experiments with model mass spectra disproved this assumption. Actually, MS Search 2.4 works differently. A mass spectrum is divided into multiple bins with a width of 1 m/z, and intensities of all peaks located inside a particular bin are summed. We believe that in the case of GC/MS data such behavior is preferable because it allows obtaining the analytical response for all isobaric ions. Our conclusion about the back end of the rounding algorithm can be checked using two model mass spectra (Fig. 3): import_ex2.msp and import_ex3.msp. Everyone can see that these mass spectra would be identical if intensities of all peaks located inside the same bin were summed. Searching these two mass spectra against any database returns the same list of candidates and identical match factors. It confirms our assumption about the rounding procedure.
Fig. 3. Importing mass spectra using the "Accurate m/z" option.
One more undocumented issue was also revealed during our experiments. It is connected with bin boundaries used during conversion to nominal mass spectra. By default, each bin extends from n - 0.5 to n + 0.5 (where n is an integer). It was found that the positions of bin boundaries can be adjusted by changing the default Spectrum Import Options (Fig. 1). At first glance, it may seem that when the Accurate m/z option is activated the value entered in the Add this term to all m/z field does not have any effect. Imported mass spectra remain absolutely the same regardless of the entered value (Fig. 4). However, it was found that the value in the Add this term to all m/z field affects the positions of bin boundaries and, as a result, the returned lists of possible candidates can be different (Fig. 4). For example, if -0.149 value were entered into the Add this term to all m/z field, bins would extend from n - 0.351 to n + 0.649. To check our conclusions everyone can use the import_ex4.msp file (which is a modification of import_ex3.msp obtained by adding 0.45 to all m/z values).
Fig. 4. Importing mass spectra using the "Accurate m/z" option and non-default value of the field "Add this term to all m/z".
In conclusion, we would not recommend using the "EI (nominal m/z)" option to import mass spectra containing floating-point m/z values, because the analytical response of some isobaric ions can be lost. As a result, intensities in the nominal mass spectrum can be distorted and results of the library search can be biased.
References:
- Mass Spectral Libraries (NIST/EPA/NIH EI & NIST Tandem 2020) and NIST Mass Spectral Search Program (Version 2.4). National Institute of Standards and Technology. Gaithersburg, 2020. 88 p.