Why MSn?

MSn in Structural Biology – Overview:

Generating high mass molecular ions from biomolecules places a great emphasis on their ordered deconstruction in the gas phase.

Unlike vibrational or nuclear magnetic resonance spectroscopy, a single stage of mass analysis (MS) provides only the mass to charge ratio (m/z) in characterizing a molecule. Measuring the m/z at high enough accuracy can in principle determine atomic composition – and this can be crucial information. However, for most analytical problems in research and industry an unknown’s atomic composition in isolation is inadequate; one needs to further characterize the relative arrangement of the atoms, i.e., the molecular structure. In biological settings the diversity of atomic species is often narrow and the molecules analyzed are commonly high-mass oligomers or polymers.

The development of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) has revolutionized the application of mass spectrometry to analytical biology. By creating gas phase ions from polar, high mass molecules, MALDI and ESI have made MS analyses of proteins, nucleic acids, carbohydrates and their various conjugates and modifications much more practical and powerful. But in substantially increasing the molecular mass available to MS analyses, these ionization methods have simultaneously decreased the significance of simply the mass in a structural characterization of the molecule. In most bioanalytical settings the dominant practical problem is to determine a specific isomeric configuration, for example, in sequencing a polypeptide or nucleic acid oligomer. Consequently, MS analyses are often carried out in concert with other analytical methods, compromising the exquisite sensitivity of MS alone.

From the inception of chemical mass spectrometry, ion fragmentation patterns associated with the ionization process have been used to identify molecules. Electron impact (EI) and chemical ionization (CI) generally give ion fragments and tables of these fragmentation patterns are a standard part of the chemical analysis of small, non polar molecules. Fast atom bombardment (FAB) is another, perhaps dated, ionization method that produces molecular fragmentation and these patterns, along with the molecular ion, have played a significant role in the identification and structural analysis of larger and more polar molecules. However, to identify molecules by ion source fragmentation, one generally requires the prior purification of the molecule. Coupling of devices for molecular isolation with EI or CI ion source fragmentation in gas chromatography mass spectrometry (GC-MS) has produced an extremely powerful analytical instrument and a correspondingly large commercial market.

In the 1980’s FAB extended MS analysis to more polar and more complex molecules. Purification of these molecules at high sensitivity was often not an option and this fueled the development of tandem mass spectrometry (MS/MS or MS2). By placing mass analyzers before and after a collision cell, ions could first be mass isolated, fragmented (by colliding the ions with neutral gas molecules or atoms), and the fragment masses analyzed. Complex mixtures could now be studied at unprecedented sensitivity. The triple quadrupole mass spectrometer is probably the best known and most successful example of the MS2 instrument.

Tandem beam instruments like the triple quadrupole suffer two fundamental defects and this has motivated interest in a number of new instrument designs. The first defect is the loss of ions at all other m/z values when transmitting ions at a given m/z value, a generic problem of beam instruments. In contrast, time of flight (TOF) mass spectrometers measure essentially every ion created – hence they generally exhibit higher sensitivity than beam instruments. The second defect of the tandem beam instrument is the limitation in the number of stages of ion isolation and fragmentation. Although instruments have been built in which greater numbers of mass analyzers and collision cells have been arranged in series, these devices, in virtue of their cost and poor sensitivity, have remained largely isolated research instruments. Ion trap devices, i.e., the Paul and Penning traps, address both of the fundamental defects of beam instruments. Like TOF, all ions created can potentially be measured. Paul and Penning traps are also capable of MSn, i.e., multiple stages of ion isolation and fragmentation. The Paul trap has been a significant commercial product for GC MS for over a decade. In the last years more capable Paul traps have been introduced with a liquid chromatography (LC) interface and an ES ion source and this device has rapidly won market acceptance.

Penning traps or, more specifically, FT ICR mass spectrometers, are also able to implement MSn techniques. One of the core research objectives of this MS Resource is to develop FT ICR applications for the structural analysis of carbohydrates. In the experimental application of FT ICR MSn to carbohydrate oligomers a number of instruments and methods were examined and in various links to this document some of this data is reviewed.

There are two themes that will be examined in this section. The first theme is the analytical utility of MSn compared with MS/MS techniques. Since the BUSM MS facility has a particular focus on carbohydrates, most of the illustrations will concern oligosaccharides and glycoconjugates. The second theme is to understand the dynamical issues behind MSn performance. Again, the BUSM MS facility has a specific mandate to investigate FT ICR applications and so much of this discussion will focus on ion dynamics in Penning traps. As the site develops I hope to incorporate more material comparing FT ICR with the Paul trap.