High‐efficiency Detection of 66 000 Da Protein Molecules Using a Cryogenic Detector in a Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometer

Abstract

We present the first experimental results obtained using a cryogenically-cooled Nb-Al,O,-Nb superconductorinsulator-superconductor (SIS) tunnel junction detector operating at 1.3 K as an ion detector in a time-of-flight mass spectrometer. As opposed to microchannel-plate ion detectors (MCPs) commonly used in such systems, cryogenic detectors such as SIS detectors offer a near 100% detection efficiency for all ions including single, very massive, slow-moving macromolecules. We describe the operating principle of an SIS detector and its use as an ion detector in our matrix-assisted laser desorptionlionization (MALDI) time-of-flight mass spectrometer and compare its response to an MCP detector operated in the same system. To our knowledge, this is the first direct comparison of these detector types in this application. A comparison of count rates and time-of-flight spectra obtained with both detectors for human serum albumin (molecular weight 66 000 Da) indicates a two to three orders of magnitude higher detection efficiency per unit area for the SIS detector at this mass. For higher molecular masses we expect an even higher relative efficiency for cryogenic detectors since MCPs show a rapid decline in detection efficiency as ion mass increases, which is not expected to be the case for cryogenic detectors.Our results imply that time-of-flight techniques could be extended beyond the current upper mass limit if cryogenic detectors are used. Initially, cryogenic detectors will be used for the analysis of large protein molecules. If non-fragmenting ionization techniques can be perfected, cryogenic detectors will also open the possibility of the rapid analysis of large DNA molecules and perhaps intact microorganisms.Cryogenic detectors' are a new class of very sensitive, energy-resolving, low-threshold particle detectors which are currently being developed for a variety of applications such as X-ray spectroscopy,2 optical ~pectroscopy,~ and searches for dark matter in the form of weakly interacting massive particles (WIMPS).~ Cryogenic detectors rely on measuring low-energy solid-state excitations as part of their detection mechanism, and therefore must be operated at temperatures typically below 2 K to avoid excess thermal excitations. The energy of these excitations, typically S 5 meV, is much less than the -eV energies needed to produce secondary electrons or electronic excitations in conventional ionization detectors. Thus a relatively large number of excitations is created for given energy deposition which allows the energy to be measured with smaller statistical error and thus much greater precision. The low excitation energy also makes cryogenic detectors more sensitive to weakly ionizing, slow moving particles than ionization detectors. Therefore, cryogenic detectors are ideal for application in time-offlight mass spectrometers used to measure the masses of large species, such as massive biomole~ules.~~~ In a typical mass spectrometer, large ions move too slowly to be efficiently detected by an ionization detecto...

Optical detection methods for mass spectrometry of macroions

“…Employing a liquid-He-cooled Sn-based semiconducting tunnel junction detector, Twerenbold et al (1996) demonstrated the concept with lysozyme (m ¼ 14.3 kDa) and other large biomolecules in a TOF-MS measurement. An Nbbased semiconducting tunnel junction detector was utilized almost at the same time by Frank et al (1996) for high-efficiency detection of human serum albumin (m ¼ 66 kDa). Single protein molecules with masses as high as 750 kDa can be detected with a signal-to-noise ratio of S/N & 5/1 (Frank et al, 1999).…”

Energy resolution improvement of superconducting tunnel junction particle detectors with infrared-blocking filters

“…The resolving power was 14 at 3 keV in this study. This value is better than the resolving power of 2-9 reported until now at higher energies of 20-30 keV [2,3,17,18]. Therefore, we have demonstrated that the infrared-blocking filter is an effective method for obtaining better energy resolution.…”

“…Recent studies have shown that the kinetic energy resolution of superconducting particle detectors is worse than that expected from X-ray measurements [2,3]. Infrared radiation is one possible source of unexpectedly bad energy resolution in superconducting particle detectors.…”

“…Because the detectors are irradiated by room-temperature radiation passing through collimators on the flight path of particles, the superconducting detectors have an operating environment that is far from ideal. To reduce the infrared radiation, simple cooled-collimators have been installed in front of superconducting particle detectors [2][3][4], however, cooling collimators do not block sufficiently incoming infrared light down the flight path of the particles in our setup. Because the subgap current of our superconducting tunnel junction (STJ) detector is degraded by 2 or 3 orders of magnitude by Cooper-pair breaking due to infrared photons or by a slight temperature increase of detector.…”

Detecting single molecules launched from liquid surfaces in a time-of-flight mass spectrometer using ultraviolet and infrared lasers

“…The protein samples are either in the form of a glycerol droplet deposited onto the sample plate or have been introduced from ambient pressure into the high vacuum of the mass spectrometer via the micronozzle device. The TOF mass spectrometer was equipped with a cryodetector for mass independent high detection sensitivity and molecule energy analyzing capability [8][9][10][11].…”