Though mass spectrometry is a century-old technique, only within the last two decades has it emerged as a tool for biomolecular analysis, particularly in the field of proteomics. A major force behind the growth of mass spectrometry-based proteomics is the combination of two fundamental technologies: TOF (time-of-flight) mass analysis and ESI (electrospray ionization).
Mass spectrometers equipped with TOF analyzers, first developed in the 1950s, separate ions according to their mass-to-charge ratio. Ions emitted from a source are accelerated so that those with like charge have the same kinetic energy. Those with a lower mass-to-charge ratio have a higher velocity than their higher mass-to-charge counterparts and thus reach the detector more quickly.
When paired with so-called hard ionization sources that break molecules into fragments during ionization, TOF analyzers have limited use in protein, peptide, and nucleic acid analysis. But, when coupled with two “soft” ionization methods that generate few fragment ions -- MALDI (matrix-assisted laser desorption ionization) and ESI (electrospray ionization) -- TOF systems can be used to analyze large biomolecules. Developed in the mid-1980s by German scientists Franz Hillenkamp and Michael Karas and Shimadzu researcher Koichi Tanaka of Japan, MALDI sources employ a chromophoric matrix in which the sample is dissolved; the matrix-sample solution is then dried on a target plate and exposed to UV laser light. The matrix absorbs energy from the laser and transfers this energy to the sample in the form of heat, which causes the sample to desorb (vaporize) and ionize.
Though MALDI-TOF mass spectrometers are common sights in most proteomics facilities, MALDI has its limitations. Most notably, MALDI is a solid-state, pulsed technique that cannot easily be coupled online with liquid-based, continuous purification methods such as HPLC. For HPLC-based applications, ESI mass spectrometers are generally the tools of choice. First described by Northwestern University scientist Malcom Dole in the 1960s, ESI gained prominence as a method for biomolecular analysis in the late 1980s through the efforts of Yale University researcher John Fenn, who earned a shared 2002 Nobel Prize in Chemistry for his work in this field.
In ESI, a liquid sample is forced through a capillary tip in the presence of an electric field. As the liquid becomes charged, its molecules begin to repel each other, forming a fine mist of charged droplets. Solvent is evaporated using a neutral carrier gas, concentrating the charged analyte molecules into smaller droplets that then explode owing to repulsive forces between like charges. The process continues until analyte ions are completely stripped of solvent and only multiply-charged ions remain. The resulting spectrum is comprised of peaks representing different charge states of the analyte. Scientists analyzing very large molecules, such as proteins and nucleic acids, cite ESI’s ability to generate these multiply-charged ions as a major advantage, as it reduces the mass-to-charge (m/z) ratio of large ions, effectively extending the mass range of the spectrometer.
ESI mass spectrometers can be coupled to an HPLC system or interfaced with a nanoelectrospray apparatus. The latter alternative reduces the high flow and sample consumption rates typical of conventional HPLC but can be labor-intensive; nano-LC systems, though they employ lower flow rates, can be complicated to use even for the most experienced technician. To address these problems, several vendors have developed novel workarounds. Agilent Technologies, for example, developed a microfluidic chip that integrates sample preparation columns with a nanospray tip that can be readily interfaced with a mass spectrometer. Additionally, Advion BioSciences last year introduced an automated nanoelectrospray system comprised of an array of nozzles that ionize individual samples from a 96-well plate and infuse them directly into an ESI mass spectrometer. And Waters Corporation recently introduced a new nanoflow LC technology featuring higher speed, throughput, and sensitivity than conventional HPLC.
A range of ESI-TOF mass spectrometers – from benchtop and LC-integrated models to complex MS “hybrids” -- are available from most of the major mass spectrometer vendors. ESI is the standard ionization source for quadrupole-TOF (Q-TOF) mass spectrometers; to increase resolution, many ESI-Q-TOF instruments feature orthogonal geometry, in which the source is perpendicular to the axis of the TOF analyzer. This configuration decouples the ionization source from the analyzer, making it possible to efficiently pair the continuous electrospray source to TOF, which operates in a pulsed mode. A sampling of commercially available ESI-TOF based systems is shown below.
References:
M. Mann et al., “Analysis of proteins and proteomes by mass spectrometry,” Annual Reviews in Biochemistry, 70:437-73, 2001.
J. M. Perkel, “Mass spectrometry applications for proteomics,” The Scientist, 15[16]:31, Aug. 20, 2001.
Brief History of Mass Spectrometry Instrumentation (Stu Borman)
http://masspec.scripps.edu/information/history/perspectives/borman.html
Introduction to Mass Spectrometry
http://masspec.scripps.edu/information/intro/index.html
Electrospray Ionization
http://poohbah.cem.msu.edu/courses/CEM924JA/Balko_Lea/ElectrosprayIonization.htm