Advanced Structural Mass Spectrometry for Systems Biology: Pulling the Needles from Haystacks

Systems-wide measurements in life sciences research have considerable promise in areas ranging from personalized medicine to understanding dominant or prevailing biological processes. However, formidable challenges remain in the comprehensive analyses of complex living systems, such as the necessity for detection limits that encompass wide concentration dynamic ranges while maintaining molecular specificity, sensitivity, and throughput. Recent work has demonstrated the ability to address several of these challenges with multidimensional separations using gas-phase ion mobility–mass spectrometry (IM-MS). IM-MS provides the capability to analyze a wide breadth of biomolecular classes on a single instrument simultaneously by separating the molecules according to their apparent surface area (equating roughly to their size) as well as their mass-to-charge ratio. The addition of a size separation parameter distributes the molecular classes into different regions of conformation space based upon differences in the prevailing intermolecular folding forces. This distribution negates the predominance of endogenous and exogenous matrix interferences experienced with traditional MS and provides a rapid (millisecond) means for separating biomolecules similar to liquid chromatography (LC)–MS approaches. Because separations are performed following ionization, additional dimensions of molecular information can be obtained through combining LC–IM-MS and gas chromatography (GC)–IM-MS. In this report, IM-MS is outlined as a separations method, several examples of the utility of IM-MS for complex biological measurements are illustrated, and the implications of this approach for systems biology research are discussed.

Systems biology seeks to describe the function of a biological system using a holistic, multiscale approach. This approach encompasses the analyses of molecular classes such as the genome, transcriptome, proteome, and metabolome (among others) of a biological system. Most modern approaches choose to either rapidly sample a single aspect (for example, the electrochemical detection of glucose and the use of green fluorescent protein as a means of monitoring translation) or infrequently sample a more comprehensive set of data. Intermittent sampling results in a fractional snapshot of the system, upon which biological inferences must be placed. When considering the turnover rate of certain enzymatically catalyzed reactions (for example, millimolar per second rates for ATP, ADP, and cytosolic glucose) or the rate of ribosomal translation (that is, six to nine amino acids per second for a eukaryotic cell), for example, single minute sampling resolution is considered coarse (1–3). The rapid acquisition rate of time-of-flight mass spectrometry (TOF-MS) measurements allows for sampling at a timescale that is relevant for a greater number of biological processes. However, MS alone lacks the necessary peak capacity for systems biology analyses, even when considering high-resolution methods that can provide a resolving power greater than ~50,000 (4,5). This limited peak capacity may be alleviated through the coupling of mass spectrometry to one of several additional separations methods, notably ion mobility (IM), which is a post-ionization separation that resolves ions based on their relative size-to-charge ratio and occurs on the order of milliseconds (6,7). Nevertheless, the ability of MS and IM-MS to perform proteomic and metabolomic analyses is unquestionable (8,9). The sensitivity, dynamic range, and ability to obtain accurate mass measurements, among other figures of merit, are the stronger suits of IM-MS.

In this article we describe the utility of IM-MS as a platform for systems biology research, the data that are acquired from these analyses, how it is interpreted, the strengths and limitations of the technology, and the outlook for future possibilities.

Theory and Instrumentation

Measuring the gas-phase electrophoretic mobility of ions, although popularized by the advances of electrospray ionization (ESI) and matrix-assisted laser desorption–ionization (MALDI) in the 1980s (10–12), really came to the forefront of separation science in the 1970s (13–17). There are currently several different forms of IM; however, all operate under similar guiding principles. Foremost, a mobility or separation cell is pressurized with a neutral background gas. The gas-phase ions from the sample are introduced into the cell and traverse the cell under the influence of a weak electric field at a rate inversely proportional to the number of collisions they experience with the background gas. Thus, smaller ions (for example, small-molecule metabolites) will traverse the cell with fewer collisions than large ions (for example, proteins). To a first approximation, these collisions are assumed to be completely elastic and the conditions of the cell (that is, electric field, temperature, and gas number density of the ion cloud) are tuned such that the collisions are of low enough energy to avoid inducing any structural or chemical changes. Thus, the elution order of ions is based upon their gas-phase packing density (that is, the physical density to which the individual molecule is folded and packed) in the gas-phase.

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