No abstract

Recent attempts to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization have claimed successful matches with more traditional methods such as size exclusion chromatography. In this paper, we describe how the resulting molecular mass distribution is not only extremely sensitive to ionization conditions such as laser power but also sensitive to the mass-and velocity-dependent detector system. For a broad polymer distribution (polydispersity > 2), employing similar ionization conditions and varying only the kinetic energy with which an ion impinges onto a detector surface, the mass corresponding to the most probable peak can be significantly shifted. Differences in shape of the measured distribution and the shift in the most probable peak are explicable by existing theories for secondary electron emission. We propose a criterion for selecting the ion kinetic energy in order to minimize spectrum distortion.

Indirect information on the conformation of highly charged molecular ions may be obtained by monitoring their collisional cross sections and the course of simple gas-phase reactions such as hydrogen-deuterium exchange. In this work, another indirect but more visually oriented approach is explored: electrosprayed protein ions are accelerated toward a highly oriented pyrolytic graphite surface and the resulting single-ion defects are imaged by scanning force and tunneling microscopy. All protein impacts generated shallow hillocks: the shapes depended on the identity and charge state of the incident protein. Lysozyme and myoglobin, both compact, globular proteins in the native state, produced compact, almost circular hillocks. However, hillocks generated by myoglobin that had been denatured in the solution phase were elongated, and the elongation was positively correlated with the charge state of the ion. It appears that structural information about gas-phase multiply charged proteins can be derived from imprints generated by energetic protein impacts on surfaces.

We present new results from an energetic surface imprinting method which allows us to outline the general conformation of protein ions in vacuo. Both disulfide-bond-intact and disulfide-bond-reduced gas-phase lysozyme ions were produced by electrospray ionization and were accelerated and impacted onto graphite surfaces. The resulting surface defects, each created by a single incident ion, were imaged with scanning force microscopy. Disulfide-intact lysozyme ions created compact, slightly elliptical hillocks on the surfaces, whereas disulfide-reduced lysozyme produced more oblong, elongated hillocks. By employing a thermal model describing the response of graphite to energy deposited by an elongated incident energetic projectile, we calculated from the hillock sizes for disulfide-reduced lysozyme (Q = 14+) an overall length of 32.1 ± 1.6 nm. This value is close to the length we observe for apomyoglobin (Q = 14+), 35.5 ± 2.4 nm, although apomyoglobin and lysozyme possess significantly different numbers of amino acid residues. Based on these results, we hypothesize that aspects of a protein's native secondary structure are preserved in the gas phase, even if the tertiary structure might be non-native. We have unfolded disulfide-intact lysozyme computationally and find a qualitatively good agreement with the experimentally obtained length of disulfide-intact (Q = 9+) lysozyme.