The long term objective of the work is to develop improved methods of identifying and characterizing biomolecules, and to apply these techniques to various biological problems. The focus of our effort is on time-of-flight mass spectrometry, (TOFMS), and in applications of mass spectrometry in proteomic and genomic research. TOF instruments have an effectively unlimited mass range and high sensitivity, so they are well suited for the examination of biomolecules. Ions are produced by electrospray ionization (ESI) or matrix-assisted laser desorption/ionisation (MALDI); both techniques can produce ions from biomolecules in the megadalton mass range. Our laboratory has constructed several TOF spectrometers for this purpose, and has been one of the pioneers in the use of """"""""orthogonal injection"""""""", where ions are introduced into the TOF instrument perpendicular to the axis. We adopted this technique first for ESI and recently introduced it for MALDI. It has demonstrated significant advantages compared to the usual axial injection method. An important contribution of our laboratory has been the use of collisional cooling of ions in a quadrupole ion guide before injection into the TOF spectrometer; such an arrangement appears to be essential for obtaining optimum results, particularly for MALDI. In addition, the laboratory has coupled a quadrupole mass analyzer (Q) and collision cell (q) to a TOF mass spectrometer to make a QqTOF instrument that is especially useful for studies of biomolecular structure. The application of collisional cooling enables MALDI ions (as well as ESI ions) to be injected into the QqTOF instrument, opening up new possibilities for measuring the structure of MALDI ions. At present this configuration is available only in our laboratory at Manitoba. Proposed applications are concerned with determining the structures of various proteins, including virus coat proteins. We are also developing methods for high throughput identification of gel-separated proteins that cannot be analyzed efficiently by conventional methods. Particular emphasis will also be placed on the study of noncovalent interactions, for which the ESI TOF instrument described above is especially useful. Various protein-ligand, protein-protein and protein-DNA complexes will be examined, and the mass limits of such measurements will be probed.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Special Emphasis Panel (ZRG1-BMT (01))
Program Officer
Edmonds, Charles G
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University of Manitoba
Zip Code
R3 2-N2
She, Yi-Min; Krokhin, Oleg; Spicer, Victor et al. (2007) Formation of (bn-1 + H2O) ions by collisional activation of MALDI-formed peptide [M + H]+ ions in a QqTOF mass spectrometer. J Am Soc Mass Spectrom 18:1024-37
Krokhin, Oleg V; Antonovici, Mihaela; Ens, Werner et al. (2006) Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis. Anal Chem 78:6645-50
Krokhin, Oleg V; Ying, Stephen; Cortens, John P et al. (2006) Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS. Anal Chem 78:6265-9
Bykova, Natalia V; Rampitsch, Christof; Krokhin, Oleg et al. (2006) Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease. Anal Chem 78:1093-103
Ozturk, Arzu; Donald, Lynda J; Li, Lin et al. (2006) Proteomic identification of AP2 gamma as a rat placental lactogen II trophoblast cell-specific enhancer binding protein. Endocrinology 147:4319-29
Krokhin, Oleg V (2006) Sequence-specific retention calculator. Algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300- and 100-A pore size C18 sorbents. Anal Chem 78:7785-95
Hadzisejdic, Ita; Cheng, Keding; Wilkins, John A et al. (2006) High-resolution mass spectrometric mapping of reovirus digestion. Rapid Commun Mass Spectrom 20:438-46
Ens, Werner; Standing, Kenneth G (2005) Hybrid quadrupole/time-of-flight mass spectrometers for analysis of biomolecules. Methods Enzymol 402:49-78
Seifers, D L; Haber, S; Ens, W et al. (2005) Characterization of a distinct Johnsongrass mosaic virus strain isolated from sorghum in Nigeria. Arch Virol 150:557-76
Donald, Lynda J; Stokell, David J; Holliday, Neil J et al. (2005) Multiple equilibria of the Escherichia coli chaperonin GroES revealed by mass spectrometry. Protein Sci 14:1375-9

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