Mass spectrometers can be smaller than a coin, or they can fill very large rooms. Although the various instrument types serve in vastly different applications, they nevertheless share certain operating fundamentals. The unit of measure has become the Dalton (Da) displacing other terms such as amu. 1 Da = 1/12 of the mass of a single atom of the isotope of carbon 12 (¹²C).

Once employed strictly as qualitative devices-adjuncts in determining compound identity-mass spectrometers were once considered incapable of rigorous quantitation. But in more recent times, they have proved themselves as both qualitative and quantitative instruments.

A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically charged ions into a proportional electrical current that a data system then reads. The data system converts the current to digital information, displaying it as a mass spectrum.

a) Increasing abundance in the total ion current (TIC) is represented as it changes over time in a chromatographic-like trace. b) Each digital slice of a peak represents the ions at that time making up the ion current often referred to as a profile or continuum acquisition. T he x or ‘time’ axis is now the mass-to-charge ratio (m/z) the ability to resolve neighboring ions in the spectrum (such as isotopes) is readily seen. c) A profile spectrum is often reduced to a ‘stick plot’ represented by centroids dropped from each peak apex reducing the size of the stored file in favor of the increased resolution information.

Ions can be created in a number of ways suited to the target analyte in question:

• By laser ablation of a compound dissolved in a matrix on a planar surface such as by MALDI
• By interaction with an energized particle or electron such as in electron ionization (EI)
• A part of the transport process itself as we have come to know electrospray (ESI) where the eluent from a liquid chromatograph receives a high voltage resulting in ions from an aerosol

The ions are separated, detected and measured according to their mass-to-charge ratios (m/z). Relative ion current (signal) is plotted versus m/z producing a mass spectrum. Small molecules typically exhibit only a single charge: the m/z is therefore some mass (m) over 1. The ‘1' being a proton added in the ionization process [represented M+H⁺ or M-H- if formed by the loss of a proton] or if the ion is formed by loss of an electron it is represented as the radical cation [M^+.]. The accuracy of a mass spectrometer or how well it can measure the actual true mass may vary as will be seen in later sections of this primer.

Larger molecules capture charges in more than one location within their structure. Small peptides typically may have two charges (M+2H⁺) while very large molecules have numerous sites, allowing simple algorithms to deduce the mass of the ion represented in the spectrum.

Low resolution instruments can deliver exceptional accurate mass when properly calibrated, but as more data crowds its limited resolution space provides less information about the spectrum. A common metabolic fragment (BK1-5 or Arg-Pro-Pro-Gly-Phe) of Bradykinin, a 9 amino acid peptide, ACE (angiotensin converting enzyme) inhibitor used to dilate blood vessels can carry two charges (single charge or M+H yields monoisotopic value 573.3149 while the doubly charged version or M+2H displays 287.1614). The isotopes are doubly charged as well begin to fill the available resolution space.

How large a molecule can I analyze?

Desorption methods (described in this primer) have extended the ability to analyze large, nonvolatile, fragile molecules. Routine detection of 40,000 Da within 0.01% accuracy (or within 4 Da) allows the determination of minor changes such as post-translational modifications. Multiple charging extends the range of the mass spectrometer well beyond its designed upper limit to include masses of 1,000,000 Da or more.

Isotope and elemental mass spectrometry

Natural isotope abundance is well-characterized. Though often thought to be stable, it can nevertheless display significant and characteristic variances. Isotope ratio measurements are used in metabolic studies (isotope-enriched elements serve as tracers) and also in climatic studies that measure temperature-dependent oxygen and carbon changes. In practice, complex molecules are reduced to simple molecular components before being measured using high-accuracy capabilities such as those found on magnetic sector instruments (see the following section).

Elemental analysis is typically performed on inorganic materials-to determine elemental makeup, not structure-in some cases using solid metal samples. Inductively coupled plasma (ICP) sources are common where a discharge (or lower power-glow discharge) device ionizes the sample. Detection using dedicated instruments, at the parts-per-trillion level, is not uncommon.