Subscriber access provided by Kaohsiung Medical University
Article
Mobility analysis of proteins by chargereduction in a bipolar electrospray source Juan Fernandez de la Mora Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Sep 2018 Downloaded from http://pubs.acs.org on September 10, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Mobility analysis of proteins by charge-reduction in a bipolar electrospray source Juan Fernandez de la Mora Yale University, Department of Mechanical Engineering and Materials Science New Haven, CT 06520-8286 USA ABSTRACT Analysis of large electrosprayed biopolymers by electrical mobility alone is greatly facilitated by reducing their charge to unity. Here we combine within a single chamber a positive aqueous electrospray (ES) producing multiply charged protein cations with a negative methanolic ES yielding small singly charged anions. Both solutions produce very small drops by including triethylammonium acetate buffer at 100 mM. The two sprays are decoupled electrostatically by an interposed 50% transparent grounded metallic grid. This screen is readily crossed by the ions, resulting in substantial charge reduction. In spite of the grid, the aqueous spray is easily destabilized by the presence of anions in the positive ES region. Nonetheless, practical ES stabilization is achieved by using relatively small capillary tips (~15 microns) in the positive emitter. Protein peaks obtained are as narrow as those previously reported via charge reduction with a radioactive Ni-63 source. Controlling the position of the negative ES permits spanning the full range of charge states, from high natural values, to predominantly singly charged ions, even for large proteins such as immunoglobulin G (~150 kDa).
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
INTRODUCTION High charge states typical of electrosprayed (ES) ions of high molecular weight solutes are essential for their analysis in mass spectrometers of limited mass range.1 High charge states also enable many diverse strategies for ion fragmentation and manipulation.2 However, the simultaneous presence of many charge states for a single species complicates the interpretation of complex spectra, particularly at high molecular weights. This difficulty is most notorious in the case of industrial polymers having broad mass distributions, 3, 4 but arises also with species acquiring many charges if the ion mass is not exactly fixed. 5, 6 For instance, clean mass spectra of viral particles with resolved peaks for neighboring charge states have been obtained only for empty capsids,7 but not for complete virions. For this reason, a variety of efforts have been directed at reducing the spectral complexity associated with variations in charge and mass. In one approach, two independent measurements are made: charge and mass/charge,8 or electrical mobility and mass/charge.9, 10 Another option is to reduce the charge state z, ideally down to unity.11, 12, 2 An early example of the usefulness of the method demonstrated the ability of a simple ion trap to select and manipulate poly(propylene imine) dendrimer ions of generation 5, 13 whose average mass (when naturally charged) could barely be guessed via high resolution ICRMS.6 Similarly, while a mass spectrometer with a resolving power of tens of thousands typically cannot mass analyze fully charged electrosprayed complete viral particles (including their DNA/RNA cargo), the same particles can be readily isolated after charge reduction by a differential mobility analyzer with a resolving power of 10.14 Because of limited m/z range, charge reduction down to z = 1 has been only rarely used by mass spectrometrists,2, 13 and typically at masses well below 100 kD. However, mobility analyzers exist that can readily analyze singly charged ions with MDa masses. For this reason, a large fraction of the published ES charge reduction studies on large species has relied on mobility instrumentation rather than MS. We have recently reviewed these studies,15 focusing on ES and charge reduction methods yielding the narrowest possible protein mobility peaks. We showed that this goal was attained by decoupling electrostatically (via a relatively transparent conducting grid) a region where proteins were electrosprayed from another region where small bipolar ions were formed by a radioactive Ni-63 source. The relatively strong electric fields produced by the spraying capillary were thus blocked by the screen, while ions of both polarities could cross the screen leading to charge reduction. The distance between the spraying tip and the screen could be made just long enough to allow complete drop evaporation, repeated Coulombic fissions and clean ion desolvation, but not overly longer, therefore avoiding unnecessary space charge dilution of the protein ions before charge reduction. In a later variant of that method, we substituted the bipolar Ni-63 source by a negative ES, also electrostatically isolated by a metal screen from the positive ES. Identical methanol solutions (~0.1-1 mM) of several ionic liquids (IL) of composition [A+B-] were sprayed from the two different polarities. Each polarity by itself produces mostly multiply charged salt clusters, but jointly electrosprayed they interact to yield dominantly singly charged cluster ions of composition [A+n-B-n±1] ±1.16 In view of the effectiveness and cleanness of the bipolar ES method to promote charge reduction, here we explore its potential when one of the sprays produces large multiply charged protein ions. As in the earlier IL study, both solutions contain the same buffer salt, triethylammonium formate (TEAF) at the same concentration (100 mM here versus ~1 mM for the ILs). The proteins are sprayed in the positive mode from aqueous solutions, while the negative spray solvent is methanol. The method has proven as effective as in the case of the ILs, though the tendency for
ACS Paragon Plus Environment
Page 2 of 9
Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
electrical discharges to form from high surface tension aqueous solutions requires the use of fairly sharp capillary tips in the positive aqueous spray.
Figure 1. Schematic of the bipolar ES chamber with two silica capillaries oriented at 90o and spraying in opposite polarities, with gas inlet (left) and outlet (right) connections. A stainless steel grid 50% transparent decouples the two emitters electrostatically (after reference 16). EXPERIMENTAL Bipolar ES chamber. This key element of the system (Figure 1) is commercialized by SEADM (Boecillo, Spain), and has been previously described.16 Briefly, a stainless steel chamber contains two silica capillaries separated by a 50% transparent stainless steel mesh. Clean gas circulates through the chamber and conveys the charge-reduced ions to a mobility analyzer. The aqueous solution fed to the positively polarized capillary includes typically 1 µM of protein with 100 mM triethyl ammonium formate. The negatively sprayed solution uses 100 mM triethyl ammonium formate in methanol. The high voltage is applied to the vial containing the solution by a stainless steel wire, and reaches the emitting tip by conduction through the liquid. In order to optimize spray quality, the aqueous solution is sprayed not far from the smallest flow rate producing a stable Taylor cone, typically with currents from 180 to 300 nA. The negative capillary is controlled to produce a spray current approximately matching that of the positive spray. The main operational difference with our prior bipolar ES study (two methanolic solutions) stems from the general difficulty to spray aqueous solutions. It is well known that water's high surface tension requires high ES voltages that tend to produce electrical discharges, which destabilize the Taylor cone. This problem may usually be handled by adding CO2 to the surrounding gas, or even in air by using sharp capillary tips with diameters in the range of 20 µm or less. However, the stability of the positive aqueous ES is considerably diminished by the negative spray, even with the screen present. Evidently, negative ions cross through the screen towards the positive tip, reach it, and disrupt the Taylor cone. We have tried a variety of methods to counteract this difficulty, including withdrawing the capillaries several mm away from the screen, (by pulling them from outside the chamber along their axes) or using large screens closing completely (without lateral gaps) the positive from the negative regions of the chamber. These approaches enable stabilizing the positive spray, but at the cost of some loss in either the level of charge reduction (resulting in charge states above z=1), or the signal of singly charged ions. The greatest flexibility in terms of capillary position and moderate screen dimensions was obtained by using relatively small tip diameters for the aqueous spray, typically below 15-20 µm. These tips were formed by pulling under a torch a capillary 360 µm and 75-180 µm in outer diameter and inner diameters (Polymicro Technologies). The closed tip of this capillary was then carefully eroded by rubbing it by hand against a polished alumina surface, until obtaining a smooth tip diameter of the desired dimensions. A tip 15-20 µm in diameter removes the spray instability, and is large
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
enough for the Taylor cone to be visually monitored with a microscope. The positive role of a small tip diameter Dt is well known in conventional (unipolar) sprays, as demonstrated by the enormous influence of the nanospray concept.17 It probably results from the reduced onset voltage Vmin required for Taylor cone formation (Vmin~Dt1/2), and the reduced dimensions of the high field region near the tip, both circumstances moderating the likelihood of negative corona ions reaching the positive tip before recombining with the positive plume. Materials: Triethylammonium formate was purchased from Fluka in 1M aqueous solution. Methanol was from Baker (ACS reagent grade). The IgG sample used was kindly provided by Drs. Michael Zachariah and Rian You from U. Maryland. It produced exceptionally narrow mobility spectra for this large protein, so a detailed description of their protocol is included in the Supporting Information. DMA/Detector. We used a previously described cylindrical Differential Mobility Analyzer (DMA)18 dubbed the Halfmini.19 It combines an axial velocity field parallel to the axes of the cylinders (driven by a blower providing a relatively high flow Rate Q of clean gas) with a radial electric field (controlled by the voltage difference VDMA between two concentric cylindrical electrodes). Ions introduced through an inlet slit in the outer electrode then move along mobility-dependent trajectories, such that only a narrow range of mobilities centered about a value inversely proportional to VDMA is extracted through an outlet slit in the inner electrode. A flow rate qi of approximately 2 Lit/min (controlled by a flowmeter) of filtered and dried air passes through the bipolar ES chamber, entraining the protein ions into the inlet slit. The mobility-selected outlet flow is captured by SEADM’s Lynx Faraday cup electrometer (
