Mass Spectra and Peak Identification

A mass spectrum is the basic form of data output by a mass spectrometer. The horizontal axis represents the mass-to-charge ratio (m/z), whilst the vertical axis represents ion abundance (intensity). A mass spectrum consists of several ‘peaks’, each of which corresponds to an ion with a specific mass-to-charge ratio; the height of a peak reflects the relative abundance of that ion. Interpreting a mass spectrum involves ‘translating’ these peaks one by one into the molecules or fragments they represent.

Peak identification involves determining which ion each peak corresponds to, based primarily on three criteria. The first is the mass-to-charge ratio value itself: the molecular ion peak directly provides the compound’s molecular weight, serving as the starting point for identification. The second is the relative intensity of isotopic peak clusters: many elements have naturally occurring isotopes that form regular companion peaks alongside the main peak—for example, when a molecule contains one chlorine atom, the ratio of m to m+2 is approximately 3:1; when a compound contains one bromine atom, the ratio of m to m+2 is approximately 3:1; and carbon-13 (¹³C) also produces a predictable m+1 peak. Based on this, it is possible to deduce which elements the molecule contains. The third is the patterns of addition and fragmentation: when identifying addition peaks such as [M+H]⁺ and [M+Na]⁺, the mass of the addend must be subtracted, whilst fragmentation peaks provide structural clues.

In mass spectrometry imaging, mass spectra are arranged according to their spatial position to form an ‘image’. Each pixel corresponds to a mass spectrum, where the intensity of a particular m/z peak represents the relative abundance of that substance at that point; by extracting the intensities of the same m/z across all pixels, one obtains a spatial distribution image of that substance. Neo Source INSTRUMENT’s MSI LDPI acquires mass spectra point by point at a resolution of 2–3 μm, whilst the MSI DPI performs the same process at a resolution of 20–200 μm—the essence of imaging is the ‘spatially-aligned assembly of a large number of mass spectra’.

The quality of peak identification directly determines the reliability of the imaging results. When mass resolution is insufficient, peaks at adjacent m/z values will overlap, potentially leading to two substances being misidentified as one, or weak peaks being masked by strong ones; when mass accuracy is inadequate, deviations in m/z positioning can result in incorrect substance identification, meaning that if a substance is mislabelled in an ‘image’, all subsequent biological interpretations will be skewed. Therefore, high-resolution, high-accuracy dual-field reflection TOF is the foundation for reliable peak identification and, consequently, credible imaging results. MSI DPI and MSI LDPI can be retrofitted onto mass spectrometers from Agilent, AB SCIEX and Thermo.