Geometric characterization of polymeric capillaries.

There are few methods in the literature to measure the inner diameter of very small capillaries. Although silica capillaries are more commonly used, synthetic polymer capillaries are preferred in some applications. The technology for producing them is not as mature. Aside from the absolute value of the inner diameter, the circularity, concentricity (a quantitative index is defined here for the first time) and the bore uniformity of such capillaries are of interest. Beyond microscopy, we describe multiple methods that determine the capillary inner diameter, averaged over a given length. The measurements variously depended on the capillary internal volume, length and cross section, and the resistance to fluid flow. The different approaches produced mutually consistent results. We show that when the internal diameter is not uniform, the different dependence on diameter that two such methods may exhibit, can be exploited to determine the true mean diameter as well as its variance. Finally, for open tubular liquid chromatography, where performance acutely depends on the inner diameter, we surprisingly find that while the mean i.d. may be the dominant determinant of efficiency, bore variance has little to no effect on the performance.

Comparison of the pH-dependent formation of His and Cys heptapeptide complexes of nickel(II), copper(II), and zinc(II) as determined by ion mobility-mass spectrometry.

The analog methanobactin (amb) peptide with the sequence ac-His1 -Cys2 -Gly3 -Pro4 -Tyr5 -His6 -Cys7 (amb5A ) will bind the metal ions of zinc, nickel, and copper. To further understand how amb5A binds these metals, we have undertaken a series of studies of structurally related heptapeptides where one or two of the potential His or Cys binding sites have been replaced by Gly, or the C-terminus has been blocked by amidation. The studies were designed to compare how these metals bind to these sequences in different pH solutions of pH 4.2 to 10 and utilized native electrospray ionization (ESI) with ion mobility-mass spectrometry (IM-MS) which allows for the quantitative analysis of the charged species produced during the reactions. The native ESI conditions were chosen to conserve as much of the solution-phase behavior of the amb peptides as possible and an analysis of how the IM-MS results compare with the expected solution-phase behavior is discussed. The oligopeptides studied here have applications for tag-based protein purification methods, as therapeutics for diseases caused by elevated metal ion levels or as inhibitors for metal-protein enzymes such as matrix metalloproteinases.

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide's mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide's acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Weak Acid-Base Interactions of Histidine and Cysteine Affect the Charge States, Tertiary Structure, and Zn(II)-Binding of Heptapeptides.

Zinc fingers are proteins that are characterized by the coordination of zinc ions by an amino acid sequence that commonly contains two histidines and two cysteines (2His-2Cys motif). Investigations of oligopeptides that contain the 2His-2Cys motif, e.g., acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, have discovered they exhibit pH-dependent Zn(II) chelation and have redox activities with Cu(I/II), forming a variety of metal complexes. To further understand how these 2His-2Cys oligopeptides bind these metal ions, we have undertaken a series of ion mobility-mass spectrometry and B3LYP/LanL2DZ computational studies of structurally related heptapeptides. Starting with the sequence above, we have modified the potential His, Cys, or C-terminus binding sites and report how these changes in primary structure affect the oligopeptides positive and negative charge states, conformational structure, collision-induced breakdown energies, and how effectively Zn(II) binds to these sequences. The results show evidence that the weak acid-base properties of Cys-His are intrinsically linked and can result in an intramolecular salt-bridged network that affects the oligopeptide properties.