Overview
Miniature Time-of-Flight Mass Spectrometer
Principal Investigator:
Richard S. Potember, Ph.D.
Organization:
Johns Hopkins University Applied Physics Laboratory
Technical Summary
Major advances must occur to protect astronauts from prolonged periods in near-zero gravity and high radiation associated with extended space travel. The dangers of living in space must be thoroughly understood and methods developed to reverse those effects that cannot be avoided. Six of the seven research teams established by the National Space Biomedical Research Institute (NSBRI) are studying biomedical factors for prolonged space travel to deliver effective countermeasures. To develop effective countermeasures, each of these teams require identification of and quantitation of complex pharmacological, hormonal, and growth factor compounds (biomarkers) in humans and in experimental animals to develop an in-depth knowledge of the physiological changes associated with space travel.
At present, identification of each biomarker requires a separate protocal. Many of these procedures are complicated and the identification of each biomarker requires a separate protocol and associated laboratory equipment. To carry all of this equipment and chemicals on a spacecraft would require a complex clinical laboratory, and it would occupy much of the astronaut's time. What is needed is a small, efficient, broadband medical diagnostic instrument to rapidly identify important biomarkers for human space exploration.
The Miniature Time-Of-Flight Mass Spectrometer Project in the Technology Development Team is developing a small, high resolution, time-of-flight mass spectrometer (TOFMS) to quantitatively measure biomarkers for human space exploration. Virtues of the JHU/APL TOFMS technologies reside in the promise for a small (less than one cubic ft), lightweight (less than 5 kg), low-power (less than 50 watts), rugged device that can be used continuously with advanced signal processing diagnostics. To date, we have demonstrated mass capability resolution from under 100 to beyond 10,000 atomic mass units (amu) in a very small, low-power prototype for biological analysis. Further, the electronic nature of the TOFMS output makes it ideal for rapid telemetry to Earth for in-depth analysis by ground support teams.
A major objective of this project was the design and development of a mass spectrometer system architecture that can be utilized for diagnostics based on complex, non-volatile biomarkers species. Because this requires multiple (and generally incompatible) ionization sources, we have designed, built and tested an orthogonal extraction time-of-flight (TOF) mass spectrometer analyzer that incorporates a daul matrix-assisted laser desorption/ionization (MALDI) and electron ionization (EI) source.
The orthogonal extraction time-of-flight instrument was successfully completed and demonstrated. This novel instrument greatly expands the spectrum of biomarkers that can be measured by incorporating the capability of electric impact ionization with the previously demonstrated MALDI measurements. This new capability allows measurement of substances ranging from low molecular volatile organic compounds to high molecular weight biological compounds such as proteins and carbohydrates.
The TOFMS Team has completed initial laboratory studies with critical biomarkers identified by the Muscle Alterations and Atrophy Team. The TOFMS Team has recorded full spectrum mass spectral signature of key target biomarker analytes using the MALDI technique at physiological concentrations found in urine. Sampling from urine has been chosen as a high priority for this project. Compounds investigated included: insulin-like growth factors (IGF-I), Urinary 3-methylhistidine, and estradiol. IGF-I is a potent anabolic factor that mimics most of the growth promoting actions of GH in vivo. IGF-1 has also been identified by the Bone Demineralization / Calcium Metabolism Team as an important biomarker.
Another biomarker identified by Muscle Alterations and Atrophy Team is urinary 3-methylhistidine. It is a measure of myofibrillar protein degradation. 3-methylhistidine cannot be re-utilized by the body. It is rapidly and quantitatively excreted in the urine. Estradiol is a steroid hormone important for the maintenance of muscle mass and bone density. It is widely speculated that steroid hormones such as estradiol play a central role in the early stages of muscle atrophy and bone demineralization.
The TOFMS team has also used matrix-assisted laser desorption mass spectrometry as a tool to quantitatively measure 3-MH in biological fluids. The TOFMS team analyzed various concentrations of 3-methylhistidine in water and in urine to determine the relationship between analyte concentration and analyte molecular ion intensity. The concentrations used in this study were based on 3-methylhistidine concentration typically found in urine, i.e. 20pmole -3.5nmole. The team examined the utility of two types of internal standards, histidine, a structural analogue, and d3-3-methylhistidine, a stable-isotope labeled analogue. 3-Methylhistidine (3-MH) samples in water and urine were prepared ranging from 5uM-10mM, keeping the (3-MH)/(histidine) ratio constant at 1:10. Protonated molecular ions for 3MH and histidine could be identified; in the corresponding MALDI spectra. A plot of the ratio of relative peak intensities of (3MH)/(d3-3-MH) versus 3-MH concentration gave a linear response with a correlation coefficient, R2=0.9799 and a relative standard deviation of the slope of 4.00 percent.
The TOFMS Team has also completed initial laboratory studies with biomarkers specific to the Bone Demineralization / Calcium Metabolism Team. These include trivalent hydroxypyridinium crosslinks and creatinine. Trivalent hydroxypyridinium crosslinks are released into the circulation during bone resorption and are excreted as free pyridinolines molecules. In bone and cartilage, the collagen is bound by pyridinoline or deoxypyridinoline crosslinks. Deoxypyridinoline is found exclusively in bone while pyridinoline is found in skin, joint and cartilage. Creatinine is used to extrapolate the status of bone remodeling activity in various metabolic bone conditions.
The TOFMS Team has performed a mass spectral analysis of alendronate to determine the mass spectral pattern by MALDI and to add the compound to our library of critical biomarkers. Bisphosphonate administration to the hindlimb of suspended rats and limb immobilization studies in dogs suggest that this compound is an effective countermeasure to bone loss. Alendronate is a member of the bisphosphonate family of drugs used to treat/prevent osteoporosis. We analyzed a commercially available product, Fosamax.
The TOFMS team has also used a breath monitoring system to examine human subjects in order to select molecules that may serve as biomarkers of normal and abnormal physiology. These molecules will be used to direct the selection of molecules to be monitored with the time-of-flight miniature mass spectrometer.
One of the objectives of the NSBRI Human Performance Factors, Sleep and Chronobiology Team is to develop strategies to monitor the circadian physiology of astronauts during long-duration space missions. The Team has identified that there is a critical need for in-flight assessment of melatonin levels. Melatonin is recognized as a very reliable marker of the human circadian pacemaker. Recent studies have indicated that there is very reliable correlation between the salivary and plasma levels. Because the sampling of plasma melatonin is an invasive procedure, it would be desirable to have a means of measuring salivary melatonin in subjects on long-duration space missions. We have performed a preliminary analysis of salivary melatonin using MALDI time-of-flight mass spectrometry of melatonin in saliva. Mass spectrometry may provide a reliable, convenient, and economical way to track melatonin during space missions.
Whole blood is the biological fluid of choice for therapeutic drug monitoring and for performing pharmacokinetic studies. Spectra for whole blood were recorded in DHB matrix and in cyano-4-hydroxycinnamic acid matrix. These spectra exhibited well-defined peaks from 100 to 400 mass units.
The risks to personnel in space from the naturally occurring radiation are generally considered to be one of the most serious limitations to human space missions. The NSBRI is examining the consequences of radiation in space in vivo in order to develop countermeasures, both physical and pharmaceutical, to reduce the risks of cancer and other diseases associated with such exposures. The consequences of exposure to radiation in space are considered a major limiting factor for long-duration interplanetary space travel for humans. Radiation doses in space may be hundreds of: times greater than those experienced on Earth. These energetically charged particles can kill cells in the body or cause mutations that may lead to cancer, cataracts, central nervous system damage or other diseases.
The TOFMS Team evaluated three novel peptide cancer biomarkers to demonstrate the utility of MALDI-TOF as a tool for the early detection of carcinomas. The advantage in using it for detection over these other methods is the robust nature of the analyzer. MALDI-TOF mass spectrometry is rapid, sensitive, and tolerant of salts in biological samples.
Summary of Satisfaction of Objectives
The first-year objectives were satisfactorily completed. In year one of the program the team prioritized the biological analytes that we would investigate. We recorded mass spectral signature data of initial biomarkers. We developed a quantification protocol to obtain biomarkers from blood, urine and breath. We coordinated TOFMS Development with other space experiments and devised a concept for a reflectron-TOFMS.
The second-year objectives were satisfactorily completed. The tasks accomplished in year two were to: build and test an electrospray sample deposition apparatus for quantitative analysis of biomarkers; evaluate and examine surfaces for sample inlet system; establish a quantitative method using isotopically labeled internal standards for specific biomarkers; develop a concept of operation for space-based processing of biomarkers from urine and blood; test the concept of operation on tabletop commercial TOFMS and DARPA "prototype" TOFMS; and, develop an orthogonal extration TOFMS design for enhanced ion collection efficiency.
The third-year objectives were satisfactorily completed. The tasks were to conduct subsystems integration; interface the system for interconnectivity and interoperability; determine TOFMS functionality; evaluate selected analytes provided by other collaborators on the TOFMS; and, evaluate analytical processing methods.
Implications of Findings
The long-term implications of this ground-based research and technology development project are to lay the scientific and engineering foundations to design, build and launch a flight-qualified "Miniature Time-of-Flight Mass Spectrometer" (TOFMS) for use on space platforms such as the Space Shuttle and the International Space Station (ISS). Successful deployment of this instrument in near-Earth missions will lay medial, engineering and scientific groundwork to adopt this medical diagnostic instrument for a mission to Mars later this century.
The development of the "Miniature Time-of-Flight Mass Spectrometer" will provide NSBRI/NASA with a complete medical diagnostic system to measure bone, internal organs, and soft tissues, routinely and non-invasively. This compact medical diagnostic system will provide autonomous and semi-autonomous patient monitoring systems with low false positive alarm rates.
This research project supports the goals of the Life Sciences Division of NASA to aid in the exploration of the solar system, support the achievement of routine space travel, and enrich life on Earth through the use of space technology and the application of biomedical knowledge. This research project falls into Category 4: Clinical Research in Support of Space Missions (Medicine in Extreme Environments).
The Countermeasure Readiness Level (CRL) developed by NASA describes the level of scientific maturity of applied research from the development of a hypothesis to validated procedure ready for operational implementation of procedures and devices. This scale has been developed as a method to mitigate the deleterious effects on humans engaged in space flight. Using this scale as a metric, this project was at level five, "Proof of concept testing and initial demonstration of feasibility and efficacy." Based on the results that we have achieved to date, we believe that this project can successfully transition to countermeasurement development.