Time-Of-Flight Mass Spectrometry

Explore the Future of Mass Spectrometry

Time-of-Flight

Robert J. Cotter (PhD) Mass Spectrometry Prof. Pharmacology and Molecular Sciences Johns Hopkins University Tools for Proteomics Research, Bioagent Detection and Diagnostics Baltimore, MD 21205, USA

he worldwide effort to (MALDI), have made it pos- Time-of-flight mass study the human pro- sible to ionize the much spectrometers larger molecules found in teome promises new In a time-of-flight (TOF) mass T biological systems, such as drug targets, improved spectrometer ions are formed in diagnostics and the ability peptides, proteins and an ion source and accelerated by DNA. Our laboratory at the potential difference (V) to predict more reliably the across the source into a drift the Johns Hopkins Universi- beneficial and adverse region. [See Fig. 1.] Typically, a responses to therapies. The ty School of Medicine has voltage of 20 kV is placed on the analytical method leading been a major player in the sample plate at the back of the source, and the ions are formed this effort is mass spectrom- development of time-of- flight mass spectrometers by a laser pulse whose duration etry, with time-of-flight may be from 1 nanosecond to as mass spectrometers and ion and their applications to little as a few picoseconds. Once traps replacing the more biological and medical in the drift region, lighter ions travel faster than heavier ones. traditional magnetic sector research. We have been fortunate in our collabora- If the ion source is small (1-2 cm) and double focusing instru- and the drift length (D) large (1 ments used for smaller mol- tion with Shimadzu Corpo- meter or more), then the total ecules. Molecules analyzed ration and their subsidiary flight time (t) of an ion is by mass spectrometry must Kratos Analytical to see the approximately the time in the incorporation of several key drift region, or t = (m/2eV)1/2D. first be ionized and two Ions of any mass can be analyzed technologies developed by new methods: electrospray by the TOF so that it is attrac- ionization and matrix- our laboratory in their bio- tive for investigating biological assisted laser analytical instruments. molecules. To improve mass res- olution, a reflectron is generally desorption/ionization

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Fig. 1 TOF mass spectrometer and innovations described by author Fig. 2 Expression and mass spectrometric analysis of antigens

used to send the ions back along and the MHC molecule as belong- the MALDI 4 and AXIMA-CFR the same drift length, expanding ing to oneself. Failure to recog- instruments and refocuses ions the difference in arrival time at nize the MHC molecule implies resulting from fragmentation in the detector between ions of dif- foreign cells and leads to rejec- the flight tube. With the addi- ferent mass and compressing the tion, as can occur following organ tion of an electronic gate for differences in time between ions or bone marrow transplantation. selecting a particular peptide of the same mass. Failure to recognize the antigen mass, amino acid sequence ions implies the presence of a tumor can be obtained for each individ- Biology drives instru- cell or a viral infection, and leads ual peptide in the mixture. In ment development: the to cell cytosis. The bound anti- collaboration with Mark Soloski gen/MHC complexes can be iso- at Johns Hopkins we have used curved-field reflectron lated by immunoprecipitation the AXIMA-CFR to characterize using an antibody to ß2 an endogenous peptide GMKFDR- Class I antigens are those derived microglobulin, and the antigens GYI derived from heat shock pro- from endogenous proteins that are released, separated by HPLC and tein. This study, carried out by displayed on cell surfaces by analyzed by mass spectrometry. Suzy Ramirez in our laboratory, major histocompatibility (MHC) Obtaining the amino acid has also identified a molecular molecules and recognized by cyto- sequences of antigens can be dif- mimic GMQFDRGYL that is toxic T-cells. [See Fig. 2.] In par- ficult using TOF mass spectrome- expressed when cells are infected ticular, the proteins are degraded try, particularly if HPLC frac- with Salmonella, and may be by proteosomes, transferred to the tions reveal a number of peptides responsible for an autoimmune endoplasmic reticulum (ER) by a in their mass spectrum. The response to self antigen following transporter protein (TAP), com- problem of sequencing mixtures infection. Thus, the CFR devel- bined with MHC and carried to provided one strong motivation oped by Timothy Cornish in our the surface of the cell. Antigens for our development of the laboratory was intended to are generally nine amino acids curved-field reflectron (CFR), a address a need in biological long, and the T-cell receptor/CD8 reflectron in which the voltage research. restricted recognition system gradient follows the arc of a cir- must recognize both the antigen cle. This reflectron was used in

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Fig. 3 Focusing ions Fig. 4 Miniature mass spectrometer over a broad mass range

Focusing all the ions: mass- The future: miniaturization microorganism. This is a com- pelling approach because genomic The AXIMA-CFR is a high perfor- correlated acceleration data (and the proteins derived mance mass spectrometer that is As a means for improving the from the genome) are likely to be being used in a high-throughput mass resolution of a TOF mass obtained far more rapidly for mode by LumiCyte (Fremont, CA) spectrometer, it is also common to microorganisms than mass spectral to characterize the biomarkers for pulse the first ion extraction libraries and are independent of disease, in particular for prostate region in the source (E1) several ionization method. Thus, time-of- cancer. Once determined, these hundred nanoseconds after the flight and other mass analyzers markers can be used diagnostically, initial laser pulse. Known vari- will continue to be useful as new and there would be a need for a ously as time-lag focusing, ionization methods are developed. miniaturized mass spectrometer delayed extraction or pulsed [See Fig. 5.] that can be located at the point of extraction, this method tends to care. An instrument for biological focus only a narrow range of agent detection would be similar. mass. Addressing the need for As bioagent markers are deter- simultaneously focusing the wide mined by high performance instru- mass ranges encompassed by par- ments, an inexpensive and lower tially-fractionated peptide mix- resolution instrument could be tures from the enzymatic diges- used to identify agents from sim- tion of proteins, mass-correlated ple molecular weight measure- acceleration (MCA) was developed ments. In work supported by the in our laboratory by Slava Kov- Defense Advanced Research Pro- toun. This method adds an addi- jects Agency (DARPA) we have Fig. 5 Biomarkers for Bg Spores tional time-dependent field (E2) developed a 3-inch TOF mass spec- in the second extraction region of trometer that has a mass range in From its early reputation as a the ion source to bring all masses excess of 60 kDaltons and a mass low mass range, low resolution into focus in a single spectrum. resolution for peptides of up to one and low duty cycle instrument, [See Fig. 3.] Mass-correlated part in 1200. [See Fig. 4.] The bio- the time-of-flight mass spectrom- acceleration is being used in our marker signals expressed for eter has emerged as a major play- laboratory by Robert English to viruses, bacteria and spores are er in proteomics, having demon- characterize biomarkers for bio- generally peptides, so that identifi- strated subfemtomole detection logical agents, and will be incor- cation by pattern recognition tech- limits for peptides, mass resolu- porated in future AXIMA instru- niques is enhanced by comparison tions of more than one part in ments.. with genomic data for each 15,000 and mass ranges into the hundreds of kilodaltons.

Robert J. Cotter is Professor of Pharmacology and Molecular Sciences at the Johns Hopkins University School of Medicine, and Director of the Middle Atlantic Mass Spectrometry Laboratory. He is currently Past President of the American Society for Mass Spectrometry and was President from 1998-2000.

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