Investigation of protein‐protein noncovalent interactions in soybean agglutinin by electrospray ionization time‐of‐flight mass spectrometry

Abstract

Noncovalent interactions in soybean agglutinin (SBA) were studied on an electrospray ionization (ESI) time-of-flight mass spectrometer constructed recently at the University of Manitoba. The high m/z range and high sensitivity of the instrument together with mild ESI interface conditions turned out to be ideal for detecting this noncovalently bonded tetrameric protein (MW approximately 116,000 Da) in low charge states (z = 23 to 27). By altering the acetonitrile content of the SBA solutions it was shown that the observed SBA tetramers are due to structurally specific noncovalent associations in solution. Octamers and dodecamers (MW approximately 350,000 Da) were also detected. Information on the quaternary structure of the tetramers was obtained by analyzing the fragment-ion spectrum resulting from the collision-induced dissociation of the tetramer ions.

Investigation of intact protein complexes by mass spectrometry

“…Although other instruments may also have extensive m/z ranges, at present the mass spectrometric analysis of biomacromolecules is largely the domain of time-of-flight (TOF) mass spectrometry (Tang et al, 1994). This is not only because of the virtually unlimited m/z range, but also because of the achievable high sensitivity and speed of TOF analysis.…”

“…Structural information can then be inferred from the resulting mass-analyzed product ions, which may be used to obtain further structural information. In the initial CID studies on protein complexes, the ESI generated ions were accelerated in the interface region between the source and the first quadrupole of the mass spectrometer (Tang et al, 1994), often termed nozzle-skimmer induced dissociation. Such experiments, however, lack proper precursor ion selection hampering the determination of the origin of the resulting fragment ions.…”

Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.

“…The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 40T and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro' residue in 40T.Electrospray ionization (ESI) mass spectrometry is fast becoming a useful tool for the study of specific noncovalent interactions involving proteins (1)(2)(3)(4)(5)22). A variety of systems have already been examined, including protein-protein, protein-ligand, enzyme-substrate, and enzyme-inhibitor complexes.…”

Structural and Functional Characterizations of the Proteasome-activating Protein PA26 from Trypanosoma brucei

“…For determination of molecular mass of the polymerized PA26 complex, the procedures employed were similar to that described previously (28,29). The experiment was performed on an electrospray ionization (ESI)-orthogonal-TOF mass spectrometer at the University of Manitoba.…”