Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor beta and derivatives.

Department of Chemistry, Oregon State University, Corvallis, USA.
Journal of the American Society for Mass Spectrometry (Impact Factor: 3.19). 04/2000; 11(3):237-43. DOI: 10.1016/S1044-0305(99)00139-7
Source: PubMed

ABSTRACT The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor beta (rhM-CSF beta) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (approximately 25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The dissociation of the multiply protonated ions of apomyoglobin ranging in charge from [M + 2H]2+ to [M + 21H]21+ have been studied using collisional activation and ion/ion reactions in a quadrupole ion trap. A variety of collisional activation conditions were explored for each charge state to determine optimal conditions for yielding the highest quality product ion spectra. Product ion spectra for charge states greater than [M + 6H]6+ were acquired using both on-resonance and off-resonance collisional activation, with on-resonance activation conditions providing the highest quality spectra. Product ion spectra for the lowest charge states could only be acquired using on-resonance collisional activation. The lowest charge states show a high propensity for losses of small molecules, as well as a number of favored amide bond cleavages, such as fragmentation C-terminal to aspartic acid residues. A novel, dominant cleavage between adjacent lysine-histidine residues was also observed, particularly for charge states higher than [M + 6H]6+. The largest number of structurally informative fragments, corresponding to b-type or y-type product ions, were produced from the intermediate charge states of [M + 10H]10+ to [M + 14H]14+. The product ion spectra for the charge states of [M + 15H]15+ and higher were dominated by the y151 ion, which appeared to be related to protonation of the N-terminus and, possibly, a secondary structure effect. The overall charge state dependent fragmentation behavior of apomyoglobin ions parallels that of other protein ions studied to date using a quadrupole ion trap in that the most extensive structural information is yielded by parent ions of intermediate charge states. This behavior is consistent with these intermediate charge states either being comprised of a diversity of parent ion structures, having a relatively high degree of proton mobility, or a combination of both.
    International Journal of Mass Spectrometry 11/2001; 212(1-3-212):359-376. DOI:10.1016/S1387-3806(01)00485-7 · 2.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dissociation of the complex between calmodulin, four calcium ions and the synthetic peptide RS20 derived from the phosphorylation site of smooth-muscle myosin light-chain kinase was studied by sustained off-resonance irradiation/collision-induced dissociation (SORI-CID) and electrospray ionisation (ESI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Gas-phase dissociation of isolated ions of calmodulin-RS20-Ca, in the 8+ charge state yielded fragments via two parallel pathways; calmodulin-Ca-4, complex ions in 5+ and 6+ charge states and RS20 ions in 2+ and 3+ charge states were detected at high resolution. The results are interpreted as indicating that RS20 binding to calmodulin in this complex is specific, involving particular non-covalent interactions that decompose readily during collision-induced dissociation. More specifically, it is proposed that the gas-phase dissociation of calmodulin-RS20-Ca-4 complex reflects the existence in vacuo of salt bridges.
    01/2001; 7(4). DOI:10.1255/ejms.449
  • [Show abstract] [Hide abstract]
    ABSTRACT: The advent of soft ionization techniques, notably electrospray and laser desorption ionization methods, has enabled the extension of mass spectrometric methods to large molecules and molecular complexes. This both greatly extends the applications of mass spectrometry and makes the activation and dissociation of complex ions an integral part of these applications. This review emphasizes the most promising methods for activation and dissociation of complex ions and presents this discussion in the context of general knowledge of reaction kinetics and dynamics largely established for small ions. We then introduce the characteristic differences associated with the higher number of internal degrees of freedom and high density of states associated with molecular complexity. This is reflected primarily in the kinetics of unimolecular dissociation of complex ions, particularly their slow decay and the higher energy content required to induce decomposition—the kinetic shift (KS). The longer trapping time of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) significantly reduces the KS, which presents several advantages over other methods for the investigation of dissociation of complex molecules. After discussing general principles of reaction dynamics related to collisional activation of ions, we describe conventional ways to achieve single- and multiple-collision activation in FT-ICR MS. Sustained off-resonance irradiation (SORI)—the simplest and most robust means of introducing the multiple collision activation process—is discussed in greatest detail. Details of implementation of this technique, required control of experimental parameters, limitations, and examples of very successful application of SORI-CID are described. The advantages of high mass resolving power and the ability to carry out several stages of mass selection and activation intrinsic to FT-ICR MS are demonstrated in several examples. Photodissociation of ions from small molecules can be effected using IR or UV/vis lasers and generally requires tuning lasers to specific wavelengths and/or utilizing high flux, multiphoton excitation to match energy levels in the ion. Photodissociation of complex ions is much easier to accomplish from the basic physics perspective. The quasi-continuum of vibrational states at room temperature makes it very easy to pump relatively large amounts of energy into complex ions and infrared multiphoton dissociation (IRMPD) is a powerful technique for characterizing large ions, particularly biologically relevant molecules. Since both SORI-CID and IRMPD are slow activation methods they have many common characteristics. They are also distinctly different because SORI-CID is intrinsically selective (only ions that have a cyclotron frequency close to the frequency of the excitation field are excited), whereas IRMPD is not (all ions that reside on the optical path of the laser are excited). There are advantages and disadvantages to each technique and in many applications they complement each other. In contrast with these slow activation methods, the less widely appreciated activation method of surface induced dissociation (SID) appears to offer unique advantages because excitation in SID occurs on a sub-picosecond time scale, instantaneously relative to the observation time of any mass spectrometer. Internal energy deposition is quite efficient and readily adjusted by altering the kinetic energy of the impacting ion. The shattering transition—instantaneous decomposition of the ion on the surface—observed at high collision energies enables access to dissociation channels that are not accessible using SORI-CID or IRMPD. Finally, we discuss some approaches for tailoring the surface to achieve particular aims in SID. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:135–167, 2005
    Mass Spectrometry Reviews 03/2004; 24(2):135 - 167. DOI:10.1002/mas.20012 · 8.05 Impact Factor

Full-text (2 Sources)

1 Download
Available from
Nov 7, 2014