Instrumentation and Ionization Methods Tutorial

Ionization Methods

FAB
Fast atom bombardment ionization (FAB or sometimes called liquid secondary ionization MS, LSIMS) is a softer ionization method than EI. The spectrum often contains peaks from the matrix, which is necessary for ionization, a few fragments and a peak for a protonated or deprotonated sample molecule. FAB is used to obtain the molecular weight of sensitive, nonvolatile compounds. The method is prone to suppression effects by small impurities. The molecular weight range is 100 to 4000 Da. Exact mass measurement are usually done by peak matching. The accuracy of the mass is the same as obtained in EI, CI.

MALDI-TOF
Matrix-assisted laser desorption (MALDI) is used to determine the molecular weight of peptides, proteins, oligonucleotides, and other compounds of biological origin as well as of small synthetic polymers. The amount of sample needed is very low (pmoles or less). The analysis can be performed in the linear mode (high mass, low resolution) up to a molecular weight of m/z 300,000 (in rare cases) or reflectron mode (lower mass, higher resolution) up to a molecular weight of 10,000. The analysis is relatively insensitive to contaminants. Mass accuracy (0.1 to 0.01%) is not as high as for other mass spectrometry methods. Recent development in Delayed Extraction TOF allow higher resolving power and mass accuracy. Some structural information for small molecules can be obtained in a "Post-Source Decay" mode, or by collisional activation.

MALDI and TOF methods Tutorial
This tutorial is based on work done at SRS ( www.srsmaldi.com ) by Gerardo Brucker, Mehrnoosh Sadeghi and Damon Barbacci.

ESI
Electrospray ionization (ESI) allows production of molecular ions directly from samples in solution. It can be used for small and large molecular-weight biopolymers (peptides, proteins, carbohydrates, and DNA fragments), and lipids. Unlike MALDI, which is pulsed, it is a continuous ionization method that is suitable for using as an interface with HPLC or capillary electrophoresis. Multiply charged ions are usually produced. ESI should be considered a complement to MALDI. The sample must be soluble, stable in solution, polar, and relatively clean (free of nonvolatile buffers, detergents, salts, etc.). Electrospray ionization is installed on the four-sector tandem instrument in Chemistry and available on two Finnigan LCQ instruments (Chemistry and Medicine) and the Finnigan TSQ 7000 (Medicine).

Electron-Capture ionization
Electron-capture (sometimes called negative ion chemical ionization or NICI) is used for molecules containing halogens, NO_2,CN, etc, and it usually requires that the analyte be derivatized to contain highly electron-capturing moieties (e.g., fluorine atoms or nitrobenzyl groups). Such moieties are generally inserted into the target analyte after isolation and before mass spectrometric analysis. The sensitivity of NICI analyses is generally two to three orders of magnitude greater than that of PCI or EI analyses. Little fragmentation occurs during NICI, and this mode of ionization is generally employed for quantitative analyses of trace amounts of compounds of known structure in conjunction with the use of heavy isotope-labeled internal standards.

Mass Spectral Analyses

Exact Mass Measurments
Exact mass measurements, sometimes referred to as "high-resolution measurements," are used for elemental-composition determination of the sample molecular ion or an ionic fragment. The basis of the method is that each element has a unique mass defect (deviation from the integer mass). The measurement is carried out by scanning with an internal calibrant (in EI or CI mode) or by peak matching (in FAB mode). The elemental composition is determined by comparing the masses of many possible compositions to the measured one. The method is very reliable for samples having masses up to 800 Da. At higher masses, higher precision or knowledge of expected composition are required to determine the elemental composition unambiguously. For the policy on the use of exact mass measurements as developed by the American Society for Mass Spectrometry, see: M. Gross, J. Am. Soc. Mass Spectrom.5(2) , 57 (1994).

GC-MS
Gas Chromatography- Mass Spectrometry permits separation of complex mixtures into single components before ionization and mass analysis. This is particularly useful when analyzing relatively low levels of target compounds derived from complex biological matrices. The target analyte must be relatively volatile or must be susceptible to conversion to a volatile derivative to permit GC separation. In general, the derivatized analyte should have a MW of less than 1000 Da in cases where GC-MS can be successfully applied. In special cases, derivatized analytes with MW 1000-2000 Da can be investigated. The ionization methods that can be used are EI and CI in positive and negative modes. This analysis is usually done at low resolving power and can be done at high resolving power for target (known) compounds for the purpose of proving compound presence.

LC-MS
Liquid Chromatography - Mass Spectrometry allows separation of complex mixtures of non-volatile compounds before introduction to the mass spectrometer. It is used extensively for compounds that have a high molecular weight or are too sensitive to heat to be analyzed by GC. The most common ionization methods that are interfaced to LC are ESI and Atmospheric Chemical Ionization (APCI) in positive and negative-ion modes. The LC is done in most cases by RP-HPLC, and the buffer system should not contain involatile salts (e.g., phosphates). ESI can be used for m/z 500-4000 and is done at low resolving power. LC-MS can be used to look at a wide variety of biologically important compounds including, peptides, proteins, oligonucleotides, and lipids.  

IRMS
Isotope Ratio MS is capable of very precise determination of ¹³ C/¹²C ratios. It is exploited principally in examining trace enrichment of ¹³C in small molecular-weight analytes (e.g. protein-derived amino acids) after biosynthetic incorporation of a ¹³C-labeled precursor. Applications of IRMS include the study of substrate disposition in humans after infusion of ¹³C-labeled precursors. IRMS can be combined with GC-MS. Other elemental ratios can also be measured; for example ²H/¹H, and ¹⁸O/¹⁶O, but ¹³C/¹²C and ¹⁵N/¹⁴N are the principal emphasis of this resource.

Fourier Transform Mass Spectrometry (FTMS)

The basis for FTMS is an ion trap (Penning cell) that allows ions formed by EI, CI, MALDI, and ESI to be accumulated and stored for time periods as long as minutes. During this time, reactions of the ions with neutral molecules can be followed. The method has the highest resolving power in mass spectrometry, a high upper mass limit, high sensitivity, nondestructive detection, and high accuracy for mass measurement. Because it uses Fourier transform detection, signal averaging and simultaneous wide-mass detection are possible. The capabilities of this instrumentation are evolving, and its potential for high-performance measurements is the highest. A Finnigan FTMS system for MALDI measurements was installed in early 1997 at the Chemistry site.