Analytical Chemistry

R.J. Cotter, "Time-of-Flight mass-spectrometry for the structural analysis of biological molecules," Analytical Chemistry, 64:1027-39, 1992. Robert J. Cotter (Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore): "

October 3, 1994

R.J. Cotter, "Time-of-Flight mass-spectrometry for the structural analysis of biological molecules," Analytical Chemistry, 64:1027-39, 1992.

Robert J. Cotter (Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore): "As mass spectrometry continues to play an increasing role in the solution of structural biology problems, the time-of-flight (TOF) mass analyzer is receiving particular attention. The method is highly sensitive and theoretically has an unlimited mass range. Scientists are able to use TOF mass spectrometry, coupled with ionization methods such as plasma desorption (PD) and matrix-assisted laser desorption/ ionization (MALDI), for rapid measuring of protein molecular weight; mass-mapping of enzymatic digests; and locating disulfide bonds, post- translational cleavages, and phosphorylation and glycosylation sites in proteins. Moreover, strategies that combine molecular weight measurements with enzyme reactions- -such as the `ladder' sequencing of peptides using amino and carboxypeptidases--have considerable appeal for those data confounded by the complex fragmentation patterns that have characterized spectra obtained from expensive, high- performance in-struments. Interest has been further enhanced by the recent availability of inexpensive, commercial instruments, including `desktop' versions of spectrometers and instruments that incorporate capabilities for comparing peptide maps with protein databases.

"TOF mass spectrometry is being used by our group to investigate the structural processing of the amyloid precursor protein (APP) and b-amyloid peptides implicated in Alzheimer's disease. Recently, we established that the insoluble 42-amino-acid amyloid peptides that form extracellular deposits (plaques) in the brain are also found in cerebral blood vessels (A.E. Roher et al., Proceedings of the National Academy of Sciences, 90:10836-40, 1993). Additionally, amyloid peptides from plaques show considerable isomerization of L-Asp to D-Asp and iso-Asp, suggesting that these peptides are `older' and may be derived from those found in the vascular region (A.E. Roher et al., Journal of Biological Chemistry, 268:3072-83, 1993). And, in other work, amino and carboxypeptidase ladder sequencing has been used to identify a TAP-dependent peptide that is recognized by alloreactive T cells specific for a Class IB antigen (C.J. Aldrich et al., Cell, in press).

"TOF mass resolution is improved by reflectrons, which compensate for kinetic energy differences in the ions being analyzed. Currently, there is considerable interest in the use of `reflectron voltage scanning' to reveal amino acid sequence fragments formed while the ions are in motion. Our latest instrumental development--the curved-field reflectron--refocuses sequence fragment ions simultaneously (T.J. Cornish et al., Rapid Communications in Mass Spectrometry, 8:781-5, 1994). By eliminating the need for scanning, it should become possible to obtain the amino acid sequences of peptides at the sub-picomolar level."


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