Patent Application
20240038517
This disclosure relates to mass spectrometry methodology, and in particular to capillary electrophoresis and interfacing techniques between capillary electrophoresis and mass spectrometry.
Mass spectrometry is one of the most information-rich analytical techniques for characterizing a broad range of samples. The past decade witnessed explosive growth in development of direct analysis and field portable mass spectrometers with the goal of bringing the analytical capability of mass spectrometry to various field applications including environmental monitoring, pharmaceutical analysis, point of care diagnosis, detection of chemical and/or biological warfare agents, forensic investigation, and discovery and research. A key component for portable mass spectrometers is an ionization source that can directly ionize the sample with minimum sample preparation and/or pretreatment. To date, numerous ambient ionization methods that allow direct sample ionization under atmospheric conditions have been reported. However, most of the existing ambient ionization methods, including desorption electrospray ionization (DESI), easy ambient sonic spray ionization (EASI), plasma-assisted desorption ionization (PADI), and direct analysis in real time (DART) require dedicated and specialized instrumentation or auxiliary gas and solvents, making them less favorable options for many field-portable mass spectrometry applications. Furthermore, complex sample preparation and pretreatment is often required. Currently, the most compelling ionization sources for portable mass spectrometers is paper spray ionization (PSI) or solid substrate-based electrospray ionization (ESI) due to their simplicity, minimal sample preparation requirements, and wide range of suitable target molecules. These techniques have been utilized in many applications including biofluid analysis, food sample analysis, and chemical reaction monitoring.
In some aspects, the techniques described herein relate to an apparatus, including: a separation capillary having an injection end and a distal end, the injection end configured to receive analyte liquid and the distal end configured to expel the analyte liquid; a sheath capillary covering the distal end of the separation capillary, the sheath capillary having a diameter that is greater than an outer diameter of the separation capillary at the distal end, the sheath capillary having a first end and a second end, the distal end of the separation capillary positioned between the first end and the second end of the sheath capillary, the sheath capillary carrying a fluid between the second end and the first end; an acoustic probe configured to vibrate positioned in contact with the at least one of the distal end of the separation capillary or the first end of the sheath capillary; and a ground terminal positioned at the first end of the sheath capillary.
In some aspects, the techniques described herein relate to an apparatus, wherein a flow rate of the fluid in the sheath capillary is less than one microliter per minute.
In some aspects, the techniques described herein relate to an apparatus, wherein a longitudinal axis of the acoustic probe is positioned between 80 degrees to 125 degrees in relation to a longitudinal axis of at least one of the separation capillary or the first end of the sheath capillary.
In some aspects, the techniques described herein relate to an apparatus, wherein the separation capillary has a length, measured between the injection end and the distal end, between 5 cm and 2 m.
In some aspects, the techniques described herein relate to an apparatus, wherein the separation capillary has a total volume of no more than 8 micro-liters.
In some aspects, the techniques described herein relate to an apparatus, wherein an inner diameter of the separation capillary is between 15 micrometers and 35 micrometers.
In some aspects, the techniques described herein relate to an apparatus, wherein a distance between the distal end of the separation capillary and the ground terminal is less than 2 millimeters.
In some aspects, the techniques described herein relate to an apparatus, wherein a voltage difference is maintained between the injection end of the separation capillary and the ground terminal.
In some aspects, the techniques described herein relate to an apparatus, wherein a flow rate of the analyte liquid in the separation capillary is between 0 nano-liters and 70 nano-liters.
In some aspects, the techniques described herein relate to an apparatus, wherein at least a portion of the inner surface of the separation capillary is coated with lipids.
In some aspects, the techniques described herein relate to an apparatus, wherein electroosmotic flow within the separation capillary is suppressed.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes small or large molecules that are positively charged, negatively charged, or neutral charged.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes cationic proteins and acidic background electrolytes.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes cationic proteins, and wherein at least a portion of the inner surface of the separation capillary has at least one of a net neutral lipid coating or a hybrid cationic lipid coating.
In some aspects, the techniques described herein relate to an apparatus, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is semi-permanent.
In some aspects, the techniques described herein relate to an apparatus, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is positively charged.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes anionic or neutral proteins and an electrolyte buffer at a pH of above 7.
In some aspects, the techniques described herein relate to an apparatus, wherein the fluid in the sheath capillary is an electrolyte with neutral pH.
In some aspects, the techniques described herein relate to an apparatus, wherein the fluid in the sheath capillary includes one or more amino acid additives.
In some aspects, the techniques described herein relate to an apparatus, wherein the one or more amino acid additives include at least one of L-serine, D-serine, or amino acid additives or L- or D-configuration.
In some aspects, the techniques described herein relate to an apparatus, including: a separation capillary having an injection end and a distal end, the injection end configured to receive analyte liquid and the distal end configured to expel the analyte liquid; an acoustic probe capillary having a probe end and an inlet end, the probe end of the acoustic probe capillary configured to vibrate and positioned in contact with the distal end of the separation capillary, the acoustic probe capillary carrying a fluid from the inlet end to the probe end; and a ground terminal positioned at the distal end of the separation capillary.
In some aspects, the techniques described herein relate to an apparatus, further including: a connector having a conduit for removably receiving the separation capillary, the connector including a slot to accommodate the acoustic probe capillary, wherein the conduit and the slot are positioned such that the probe end of the acoustic probe capillary makes contact with the distal end of the separation capillary.
In some aspects, the techniques described herein relate to an apparatus, wherein the connector further includes a housing to house a ground terminal, wherein the housing and the conduit are positioned to such that the ground terminal makes contact with the distal end of the separation capillary.
In some aspects, the techniques described herein relate to an apparatus, wherein a portion of the acoustic probe capillary is coupled with a vibrating structure.
In some aspects, the techniques described herein relate to an apparatus, wherein the vibrating structure includes a piezoelectric transducer.
In some aspects, the techniques described herein relate to an apparatus, wherein a flow rate of the fluid in the acoustic probe capillary is less than one microliter per minute.
In some aspects, the techniques described herein relate to an apparatus, wherein the separation capillary has a length, measured between the injection end and the distal end, between 5 cm and 2 m.
In some aspects, the techniques described herein relate to an apparatus, wherein the separation capillary has a total volume of no more than 8 micro-liters.
In some aspects, the techniques described herein relate to an apparatus, wherein a voltage difference is maintained between the injection end of the separation capillary and the ground terminal.
In some aspects, the techniques described herein relate to an apparatus, wherein an inner diameter of the separation capillary is between 15 micrometers to 35 micrometers.
In some aspects, the techniques described herein relate to an apparatus, wherein a distance between the distal end of the separation capillary and the ground terminal is less than 2 millimeters.
In some aspects, the techniques described herein relate to an apparatus, wherein a flow rate of the analyte liquid in the separation capillary is between 0 nano-liters and 70 nano-liters.
In some aspects, the techniques described herein relate to an apparatus, wherein at least a portion of the inner surface of the separation capillary is coated with lipids.
In some aspects, the techniques described herein relate to an apparatus, wherein electroosmotic flow within the separation capillary is suppressed.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes small or large molecules that are positively charged, negatively charged, or neutral charged.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes cationic proteins and acidic background electrolytes.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes cationic proteins, and wherein at least a portion of the inner surface of the separation capillary has at least one of a net neutral lipid coating or a hybrid cationic lipid coating.
In some aspects, the techniques described herein relate to an apparatus, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is semi-permanent.
In some aspects, the techniques described herein relate to an apparatus, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is positively charged.
In some aspects, the techniques described herein relate to an apparatus, wherein the analyte liquid includes anionic or neutral proteins and an electrolyte buffer at a pH of above 7.
In some aspects, the techniques described herein relate to an apparatus, wherein the fluid in the acoustic probe capillary is an electrolyte with neutral pH.
In some aspects, the techniques described herein relate to an apparatus, wherein the fluid in the acoustic probe capillary includes one or more amino acid additives.
In some aspects, the techniques described herein relate to an apparatus, wherein the one or more amino acid additives include at least one of L-serine, D-serine, or amino acid additives or L- or D-configuration.
In some aspects, the techniques described herein relate to a connector, including: a connector body having a first end and a second end; a conduit extending between the first end and the second end of the connector body, the conduit configured to removably receive a separation capillary, wherein a portion of the conduit at the first end of the connector body accommodates a distal end of the separation capillary; and an acoustic probe slot positioned at the first end of the connector body, the acoustic probe slot extends between an outside surface of the connector body and the inner surface of the conduit, the acoustic probe slot configured to removably receive an acoustic probe having a sheath capillary.
In some aspects, the techniques described herein relate to a connector, further including: a ground terminal slot positioned at the first end of the connector body, the ground terminal slot extends between an outside surface of the connector body and an inner surface of the conduit, ground terminal slot configured to removably receive a ground terminal.
In some aspects, the techniques described herein relate to a connector, further including: A dead stop positioned in the conduit at the first end of the connector body, the dead stop configured to have a diameter that is less than a diameter of the separation capillary such that the separation capillary is prevented from being pushed out of the conduit at the first end.
In some aspects, the techniques described herein relate to a connector, wherein the connector body is formed of at least one of metal or thermoplastic.
In some aspects, the techniques described herein relate to a connector, wherein the connector body includes a dead stop formed on an inner surface of the conduit, wherein the dead stop has a diameter that is less than the diameter of the separation capillary.
In some aspects, the techniques described herein relate to a connector, wherein the connector body includes: a nut portion and a port portion, wherein the nut portion screws into the port portion, wherein a portion of the conduit is formed in the nut portion and a second portion of the conduit is formed in the port portion, and a dead stop formed on the inner surface of the second portion of the conduit in the port portion, wherein the dead stop has a diameter that is less than the diameter of the separation capillary.
In some aspects, the techniques described herein relate to a connector, further including; a ground terminal slot positioned at the first end of the connector body, the ground terminal slot extends between an outside surface of the connector body and an inner surface of the conduit, ground terminal slot configured to removably receive a ground terminal, wherein the ground terminal slot is formed in the port portion.
In some aspects, the techniques described herein relate to a connector, wherein the acoustic probe slot is formed in the port portion.
Like reference numbers and designations in the various drawings indicate like elements.
The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.
Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.
While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.
It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.
It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.
As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a proton beam degrader,” “a degrader foil,” or “a conduit,” includes, but is not limited to, two or more such proton beam degraders, degrader foils, or conduits, and the like.
The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
Coupling capillary electrophoresis (CE) to mass spectrometry (MS) is a powerful strategy to leverage a high separation efficiency with structural identification. Traditional CE-MS interfacing relies upon voltage to drive this process. Additionally, sheathless interfacing requires that the electrophoresis generates a sufficient volumetric flow to sustain the ionization process. Vibrating sharp-edge spray ionization (VSSI) is a new method to interface capillary electrophoresis to mass analyzers (VSSI is discussed in further detail in U.S. Pat. No. 11,600,481, which is incorporated by reference herein in its entirety). In contrast to traditional interfacing, VSSI is voltage-free, making it straightforward for CE and MS. New nanoflow sheath CE-VSSI-MS is introduced herein to reduce the reliance on the separation flow rate to facilitate the transfer of analyte to the MS. The nanoflow sheath VSSI spray ionization functions at less than 1 microliter per min, or for example from 400 to 900 nL/min. Using the nanoflow sheath discussed herein, volumetric flow rate through the separation capillary is less critical, allowing the use of a small (e.g., 15 to 35 μm or 20 to 25 μm) inner diameter separation capillary and enabling the use of higher separation voltages and faster analysis. Moreover, the use of a nanoflow sheath enables greater flexibility in the separation conditions. The nanoflow sheath can be operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. This includes the use of a wider pH range as well. The versatility of nanoflow sheath CE-VSSI-MS is demonstrated by separating cationic, anionic, and zwitterionic molecules under a variety of separation conditions. The detection sensitivity observed with nanoflow sheath CE-VSSI-MS is comparable to that obtained with sheathless CE-VSSI-MS as well as CE-MS separations with electrospray ionization interfacing. A bare fused silica capillary can be used to separate cationic β-blockers with a near-neutral background electrolyte at concentrations ranging from 10 nM to 5 μM. Under acidic conditions, 13 amino acids are separated with normal polarity at a concentration ranging from 0.25 to 5 μM. Finally, separations of anionic compounds are demonstrated using reversed polarity under conditions of suppressed electroosmotic flow through the use of a semipermanent surface coating. With a near-neutral separation electrolyte, anionic nonsteroidal anti-inflammatory drugs are detected over a concentration range of 0.1 to 0.5 μM.
Mass spectrometry provides critical information about and bioanalytical research, especially when combined with liquid chromatography separations to reduce the sample complexity prior to mass analysis (Ref. 1-1). Although liquid chromatography is a prevalent separation method coupled to MS, capillary electrophoresis is an alternative liquid-based separation method that has been successfully interfaced to MS through electrospray ionization. Capillary electrophoresis offers the advantages of low volume sample requirements, automation, and fast runs (Ref 1-2). These features make an integrated capillary electrophoresis MS system a powerful technique for metabolomics (Refs. 1-3-1-7), proteomics (Refs. 1-8-1-9), glycomics (Ref 1-10), biomarkers (Refs. 1-11-1-14), and affinity binding (Refs. 1-15-1-16). Notable applications of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) include analyses at the cellular level (Refs. 1-3, 1-4, 1-7-1-9) as well as pharmaceutical determinations ranging from purity, degradation, and metabolite studies (Ref 1-17). Progress in the coupling of capillary electrophoresis and MS continues to advance applications that integrate these methods (Ref 1-18).
Electrospray ionization is interfaced directly to the mass analyzer as a sheathless flow, or it includes an additional sheath flow to assist in the electrospray process. The incorporation of a sheath flow offers greater flexibility in the design of the capillary electrophoresis separation. This is particularly important under conditions of suppressed or reversed electroosmotic flow in which there is little or no bulk fluid flow exiting the separation capillary. Although the use of a sheath flow sustains the electrospray process, it also dilutes the analyte band exiting the separation capillary. This process of analyte dilution can be minimized by reducing the volumetric flow rate of the sheath to nanoliters per minute. In a recent comparison of sheathless electrospray ionization to nanoflow sheath electrospray ionization, the addition of the sheath demonstrated a 10- to 100-fold dilution factor depending on the analyte (Ref 1-19). Different strategies are reported to achieve nanoliter/minute flow rates (Ref. 1-18). The use of a sheath flow of 400 nL/min19 or lower (Ref 1-20) was achieved by splitting the fluid flow delivered by a syringe pump operated at 4 μL/min. With a 3D printed plug-and-play design, the separation capillary and sheath capillary are positioned in a way that prevents back-flow into the separation capillary (Ref 1-20). A second design integrated in a commercial instrument (Ref 1-21) delivers a nanoflow sheath reported to be 50 nL/min22 through electrokinetic pumping of fluid.
Vibrating sharp-edge spray ionization (VSSI) is an alternative method of interfacing liquid separations to MS. VSSI achieves sample nebulization and ionization through the generation and application of acoustic waves through a sharp edge coupled to a piezoelectric transducer. The introduction of acoustic waves to the liquid surface results in a plume of droplets that desolvate to produce gas phase ions suitable for MS analysis. Different designs couple the acoustic energy and liquid through the sharp edge, including the corner of a piece of glass for direct contact-based nebulization (Ref. 1-23-1-24) or using a capillary attached to the piece of glass (Ref. 1-25-1-29) with fluid through the capillary. Unlike traditional electrospray interfacing, VSSI is voltage-free ionization and does not utilize nebulizer gas. Organic additives, which are commonly used to assist electrospray ionization interfacing, are compatible with VSSI and decrease droplet size.23 VSSI is compatible with fluid flow rates from 5 to 50 μL/min when the liquid passes through the probe in a design similar to what is observed with electrospray systems (Ref 1-23, 1-25, 1-27). Lower flow rates have been coupled to VSSI with field (Ref 1-26) or when liquid is brought into contact with the tip of the glass VSSI probe as was previously reported in a sheathless capillary electrophoresis-VSSI-mass spectrometry (CE-VSSI-MS) design (Ref 1-30). In that report, for the first time, a hollow pulled probe was used on the corner of a glass slide attached to the piezoelectric transducer and functioned with flow rates ranging from 70 to 200 nL/min.
Some sheathless CE-VSSI-MS systems demonstrate a limit of detection of 2 nM (Ref 1-30) which was comparable to limits of detection of 0.7 nM for beta blockers obtained with a sheathless CE-ESI-MS system (Ref 1-31). Sheathless CE-ESI-MS interfacing can be ideal for low detection limits because it avoids analyte dilution. For example, the introduction of a nanoflow sheath to CE-ESI-MS systems increased the limit of detection to 70 nM (Ref. 1-32). While the sheathless CE-VSSI-MS system was capable of detecting nanomolar levels of analyte (Ref. 1-30) separation conditions were limited to the use of a 50 μm inner diameter separation capillary and background electrolyte systems that maintained an electroosmotic flow sufficient to sustain the VSSI process.
These challenges are overcome using a sheath flow CE-VSSI-MS operated at sub-microliter per minute flow rates (e.g., 400-900 nL/min) regardless of the separation flow rate. The sheath flow can considerably reduce the requirements for an electroosmotic flow, and it enables the use of a smaller bore capillary. The use of a smaller inner diameter reduces the effects of siphoning and allows for the application of a higher separation voltage, which reduces the run time. This modular design makes the VSSI probe and capillary independent so that either is easily replaced. With the probe design, the interaction of the fluid and the sharp edge directs the nebulized plume in an angular fashion. The nanoflow sheath design is capable of detecting 10 nM pindolol with a separation efficiency of 50,000 plates per meter.
The functionality of the nanoflow sheath CE-VSSI-MS system is evaluated under different conditions to demonstrate the flexibility of this technique. The nanoflow sheath is operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. Using bare-fused silica, CE-VSSI-MS separations of β-blockers are achieved using an ammonium acetate background electrolyte at pH 6.3 that provides an electroosmotic flow. The capillary electrophoresis separations are compatible with electrokinetic injections and electrokinetic stacked injections to achieve a linear range of 10 to 1000 nM and 1 to 100 nM, respectively. Additionally, sheath flow CE-VSSI-MS can be performed where the electroosmotic flow is suppressed using a 2% formic acid background electrolyte to separate and detect amino acids within a concentration range of 0.25 to 5 μM. Moreover, the electroosmotic flow is also suppressed using a semipermanent coating to separate anionic nonsteroidal anti-inflammatory drugs (NSAIDs). The separation is achieved at near-neutral pH with reversed polarity. Under these operating conditions, NSAIDs are detectable within a concentration range of 0.1 to 0.5 μM using electrokinetic sample stacking. The detection of the anionic analytes with VSSI-MS is achieved with positive mode MS, which demonstrates a 3 order of magnitude enhancement in signal relative to that observed in negative mode MS.
Materials and Methods
Materials and methods described herein are specific examples introduced merely in relation to demonstrations and experiments discussed below and should not be viewed as limiting the scope of the claims.
Chemicals and Reagents. An amino acid kit (LAA-21), pindolol (P-0778), acebutolol (A-3669), atenolol (A-7655), timolol (T-6394), tolmetin (T-6779), ketoprofen (K-1751), suprofen (S-9894), indoprofen (I-3132), acetic acid (A6283), and mineral oil (M5904) were purchased from Millipore Sigma (Burlington, MA). Oxprenolol (156023) was purchased from ICN Biomedicals Inc. (Aurora, OH). Caffeine (C5-3) was purchased from Aldrich Chemical Co. LLC (Milwaukee, WI). Formic acid (A13285) and ammonium acetate (A16343) were purchased from Alfa Aesar (Heysham, England). Ammonium hydroxide (BDH3016) was purchased from VWR Analytical (Radnor, PA). The phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were from Avanti Polar Lipids (Alabaster, AL). Deionized water (18 MΩcm) was obtained from an Elga Purelab and Veolia Chorus water system (Lowell, MA).
VSSI Probe Fabrication. The bare fused silica sheath capillary with an inner diameter of 200 μm and outer diameter of 363±10 μm (TSP200350, Molex, Phoenix, AZ) is cut to 30 cm. The polyimide coating is removed on both ends of the capillary and examined with a microscope for a clean cut. To prepare the capillary electrophoresis electrode, a 25 μm platinum wire (PT005113, Goodfellow, Huntingdon, England) is trimmed to approximately 1 cm in length, bent at two 90° angles in a “U” shape, and fixed to the sheath using 1 minute epoxy (1366072, Henkel, Dusseldorf, Germany). To create the electric connection, the platinum is attached to a wire using conductive epoxy (8331-14G, MG Chemicals, Ontario, Canada) and then covered with a layer of 1 minute epoxy (1366072, Henkel). The hydrodynamic flow through the sheath capillary is driven by applying a pressure of 20 to 40 kPa (3 to 6 psi). The VSSI probe device is constructed using a solid glass rod (GR100-4 Precision Instruments 1 mm×10 cm), pulled using a laser puller (Sutter Instrument Company, Novato, CA), trimmed to a final tip diameter of approximately 60 to 100 μm, and attached to the underside of a piezoelectric transducer (7BB-27-4 LO, Murata diameter=27 mm) using 5-minute epoxy. The parameters used for pulling the glass rod to form the VSSI probe are as follows: HEAT=750, FIL=4, VEL=60, DEL=130, and PUL=50.
VSSI Instrumentation. The piezoelectric transducer is connected to a function generator (DDS signal generator/counter Koolertron, Hong Kong Karstone Technology Co, Hong Kong) and amplifier (7500, Krohn-Hite, Brockton, MA), and a square wave is applied with frequencies ranging from 92 to 96 kHz and amplitudes ranging from 10 to 12 Vpp. The applied frequency and amplitude vary between fabricated devices, and optimal settings are evaluated prior to use through visual observation of a microdroplet plume via a moistened cotton swab tip. A micromanipulator is utilized to position the VSSI probe at a 90° angle relative to the capillary. The VSSI probe is parallel to the platinum electrode at the end of the capillary. The sheath flow VSSI device is placed 2 to 3 mm from the inlet of the mass spectrometer. The alignment is evaluated by direct infusion of a standard compound (e.g., pindolol).
CE-VSSI-MS Separation and Analysis. Unless otherwise noted, the dimensions of the separation capillary are 30 cm, 25 μm inner diameter, and 150 μm outer diameter (TSP050150, Molex, Arizona, USA). Injection conditions, electric field, and separation polarity are noted in the text and figure captions. The acoustic spray is off during extensive pre-run flushes but remains active during shorter flushes in between runs. For the separations of beta blockers, prior to use, the capillary is flushed at 138 kPa (20 psi) with 0.1 N ammonium hydroxide for 60 min, water for 10 min, and the background electrolyte for 30 min. In between runs, the capillary is flushed for 1 min with background electrolyte. For the separations of amino acids, prior to use, the capillary is flushed at 138 kPa (20 psi) with 2% formic acid for 20 min. In between runs, the capillary is flushed for 1 min with background electrolyte. For the separations of NSAIDs, the capillary is treated with a semipermanent lipid coating. The procedure for coating the capillary involves a flush at 172 kPa (25 psi) with 0.1 N ammonium hydroxide for 30 min, deionized water for 15 min, methanol for 15 min, deionized water for 15 min, 50 mM ammonium acetate at pH 6.3 for 3 min, phospholipid coating for 20 min, and 50 mM ammonium acetate at pH 6.3 for 7 min. For all separations, the capillary used to split the flow for the sheath is 50 cm with an inner diameter of 200 μm (TSP050375, Molex, Arizona, USA), and the same background electrolyte solution is used. A Q-exactive mass spectrometer equipped with LTQ Tune Plus software (version 2.7) is used to collect the data (Thermo Fisher Scientific, San Jose, CA). Both the MS ion transfer tube and the capillary outlet are grounded, creating a field-free region. The instrument is operated with the VSSI source by overriding two interlocks for the standard HESI source and with the sweep gas cone removed. Data are processed using Thermo Fisher Scientific Xcalibur (version 4.1) and Microsoft Excel (2021, Microsoft, Redmond, WA).
Capillary Electrophoresis-UV (CE-UV) Absorbance Detection Separation and Analysis. All separations are conducted using a Beckman/Coulter P/ACE MDQ (Beckman Coulter, Fullerton, CA, USA). Capillaries have a total length of 40 cm, an effective length of 30 cm, an inner diameter of 25 μm, and an outer diameter of 360 μm. The capillary preparation and separation are the same as those used for the VSSI-MS analyses. To maintain the same electric field strength used on the CE-VSSI-MS system, the separation voltage was 21.3 kV, applied in normal polarity for the beta blocker and in reverse polarity for the NSAIDs, and 16 kV, applied in normal polarity for the amino acids. The analyses were performed using UV absorbance detection at a wavelength of 200 nm. The cartridge temperature is set to 25° C. for flushes and separations. Data collection and analysis are accomplished by the accompanying Beckman/Coulter P/ACE MDQ 32Karat Software (Beckman Coulter). It is worth noting that, in the CE-UV NSAID separations, capillaries are flushed in between runs for an additional 3 min with ammonium acetate, followed by 5 min of phospholipid, and then 3 min of background electrolyte at 172 kPa (25 psi).
Preparation of Phospholipid. The solutions of the phospholipids were prepared as previously described (Refs. 1-33-1-35). Briefly, DMPC and DHPC are weighed and combined at a molar ratio of 0.5 DMPC/DHPC. The 5% phospholipid solution is prepared by adding 50 mM, pH 6.3 ammonium acetate to the phospholipid powder. The preparation was then vortexed until the solids were thoroughly dissolved. The solution was then subjected to three freeze-thaw cycles followed by centrifugation at 10000 rpm for 10 min at 4° C. and stored at −20° C. Before use, calcium chloride was added to the lipid to a final concentration of 1 mM.
Results and Discussion
VSSI Interface Design. The VSSI interface design integrates a sheath flow that allows for stable VSSI nebulization and ionization. The sheath flow VSSI interface shown in
The separation capillary 104 can have a length, measured between the injection end 102 and the distal end 114, between about 5 cm and about 2 m. In some examples, the length of the separation capillary 104 can be between about 20 cm and about 2 m. In some examples, the inner diameter of the separation capillary 104 is between about 15 micrometers and about 35 micrometers, or about 10 micrometers to about 50 micrometers or about 10 micrometers to about 100 micrometers. In some examples, the separation capillary 104 can have a total volume of no more than about 8 micro-liters. In some examples, the separation capillary 104 can have a total volume of no more than 2 micro-liters. The total volume of the separation capillary 104 can be a function of the inner diameter and the length. As an example, the total volume of the separation capillary can be about 200 nano-liters. The distal end 114 of the separation capillary 104 can be positioned inwards from the first end 116 of the sheath capillary 112. Positioning the distal end 114 inwards from the first end 116 of the sheath capillary 112, can help in mixing the analyte liquid 108 exiting from the distal end 114 with the sheath fluid surrounding the sheath capillary 112 before the mixture is ionized by the acoustic probe. In some examples, the distance between the distal end 114 of the separation capillary 104 and the first end 116 of the sheath capillary 112 is less than 2 mm. In some examples, the distance between the distal end 114 of the separation capillary 104 and the first end 116 of the sheath capillary 112 can be less than 100 micrometers.
A ground terminal 124 can be positioned at the first end 116 of the sheath capillary 112. In particular, the ground terminal 124 can be positioned outside of the sheath capillary 112 at the edge of the first end 116. In some examples, the ground terminal 124 can be positioned at the first end 116 of the sheath capillary 112 but inside the sheath capillary 112. This can be done to position the ground terminal 124 closer to the distal end 114 of the separation capillary 104. A high voltage terminal 110 can be positioned in contact with the analyte liquid 108 in the analyte reservoir 106. A voltage difference between the high voltage terminal 110 (at the injection end 102) and the ground terminal 124 (at the distal end 114) can help maintain a flow rate of the analyte liquid 108 through the separation capillary 104. The high voltage terminal 110 and the ground terminal 124 can be coupled with a DC voltage source (not shown) that can provide a voltage difference of at least a few hundred volts and up to several hundred kilovolts. As an example, the flow rate of the analyte liquid 108 through the separation capillary 104 can be about 0 nano-liters to about 70 nano-liters.
An acoustic probe 128 configured to vibrate is positioned in contact with at least one of the first end 116 of the sheath capillary 112 or the distal end 114 of the separation capillary 104. The acoustic probe 128 can be coupled with a vibrating medium that provides vibrational motion to the acoustic probe 128. The vibrations caused by the acoustic probe 128 can cause the mixture of the stealth fluid and the analyte liquid 108 to nebulize and ionize and form a spray 126 that is directed towards the MS. In some examples, the acoustic probe 128 may be in indirect contact with the at least one of the first end 116 of the sheath capillary 112 or the distal end 114 of the separation capillary 104. That is, the acoustic probe 128 can be coupled with the sheath capillary 112 or the separation capillary 104 by an adhesive or binding material such as, for example, epoxy or polymer.
VSSI Droplet Formation. In some examples, the capillary electrophoresis-VSSI spray 126 is ejected at an angle and creates droplets that are approximately 8 μm in diameter. This angular direction of spray observed with sheath flow VSSI (
Separation Performance. Thorough characterization of the nanoflow sheath system with beta blockers is performed using electrokinetically stacked injections and the near-neutral pH background electrolyte, which has an intermediate electroosmotic flow rate. The traces for both the CE-UV and CE-VSSI-MS, shown in
Stacking Enhancement. Nanoflow sheath (e.g., the sheath capillary 112) is compatible with both electrokinetic injections (
CE-VSSI-MS Separations of Amino Acids with Suppressed Electroosmotic Flow. CE-VSSI-MS is not only useful for the separation of cationic beta blockers but also applicable to compounds that require an acidic background electrolyte to remain cationic. The separation of amino acids is performed using a background electrolyte composed of 2% formic acid, similar to CE-ESI-MS separations of amino acids reported in the literature (Ref 1-37). Under these conditions, the electroosmotic flow is suppressed; therefore, the analyte migration is predominantly due to electrophoretic mobility. The sheath design enables MS detection of analytes regardless of the electroosmotic flow rate. With CE-VSSI-MS, 13 amino acids are separated and detected in under 5 min. The separation of amino acids shown in
CE-VSSI-MS vs CE-UV. The separation performance with CE-VSSI-MS of amino acids can be compared using separations obtained with the same conditions but detected with UV absorbance detection. For CE-UV, only 7 of the 13 amino acids are detected with an electrokinetic stacking injection at 10 kV for 4 s. Also, because of peak overlap with CE-UV, full visualization of analyte peaks with UV requires two separate runs, which are then superimposed as shown in
CE-VSSI-MS Separations with a Modified Capillary Surface and Reversed Polarity. The flexibility of the nanoflow sheath CE-VSSI-MS enables the use of reversed polarity with a suppressed electroosmotic flow to resolve the anionic NSAIDs. The passivation of the inner capillary wall surface of the separation capillary 104 can be accomplished using a previously reported semi-permanent capillary surface coating (Refs. 1-42-1-44) that masks the surface charge on the fused silica through the self-assembly of a phospholipid and is stable in solutions ranging in pH from 4 to 8. A benefit of the use of this reversed polarity separation is that it enables sample stacking of anions, which increases the peak height and area. The CE-VSSI-MS analysis of NSAIDs shown in
The CE-VSSI-MS separation of NSAIDs is obtained in a single run (
The apparatus and methods described herein provide aspects of a nanoflow sheath CE-VSSI-MS design. Using this design, the separation can be performed using background electrolyte at different pH values regardless of flow rate in such a manner that high separation voltage and rapid separation can be achieved. The results achieved by this system are comparable to those achieved by the previously reported sheathless CE-VSSI-MS, and analyte is detected without a significant dilution effect. The nanoflow sheath design offers the flexibility to select different volatile background electrolytes for the separation and analysis of small cationic, zwitterionic, and anionic compounds. Moreover, separation of these analytes via the nanoflow sheath is achieved with an untreated fused silica capillary as well as a capillary surface modified with a semipermanent lipid coating. Signal enhancement is realized by the stacked injection of the analytes.
Protein Analysis Using CE-VSSI-MS
Capillary electrophoresis interfaced to mass spectrometry with electrospray ionization typically incorporates additives that are acidic or comprised of organic solvents to assist in the ionization process. Vibrating sharp-edge spray ionization (VSSI) is a voltage free method of ionization to interface capillary electrophoresis and mass spectrometry that does not require these additives, making it more suitable for a wide range of protein analyses. Additionally, when a nanoflow sheath capillary electrophoresis system is interfaced to the mass spectrometer, the separation can be performed under low and even zero flow conditions. In this report, a nanoflow sheath and capillary separation are performed with aqueous solutions and interfaced to mass spectrometry through VSSI. To accomplish these separations semi-permanent coatings comprised of self-assembled lipids are used to overcome surface adsorption and to facilitate electrophoresis at a neutral pH. Using low ionic strength aqueous solutions in the sheath fluid reduced signal suppression while including serine in the sheath fluid reduced analyte adduction. Capillary electrophoresis-VSSI separations of anionic proteins are easily detected in the 2.5-10 μM range. The anionic proteins β-lactoglobulin and transferrin are resolved using an unmodified fused silica separation capillary because they do not exhibit non-specific surface adsorption. Conversely, separations of cationic proteins cytochrome c, ribonuclease A, and α-chymotrypsinogen A are more difficult to achieve, owing to non-ideal surface interactions. With an unmodified capillary, the cationic protein separations required acidic background electrolytes to reduce electrostatic interactions. Alternatively, separations of cationic proteins are achievable at neutral pH with semi-permanent coatings based on a self-assembled zwitterionic lipid coating as well as a hybrid cationic lipid coating. These results demonstrate the potential of capillary electrophoresis-VSSI as a biotechnology tool to support protein analyses under physiologically relevant conditions.
Capillary electrophoresis (CE) technologies are powerful tools for analyses of protein therapeutics, (Ref 2-1, 2-2) protein interactions, (Ref 2-3) and can provide insight into complex protein systems that are foundational to life processes. (Ref 2-4). The simplest analyte separations are based on differences in the charge-to-size ratio (i.e. free solution electrophoresis) or on size-based sieving through gel matrices. In addition to these separation modes, CE analyses can be modified to provide information about protein activity and protein affinity. (Ref. 2-5). CE separations are exceptionally fast.6 These separations are critical to nano and microscale biological applications because approximately 1 nL of sample is consumed. The sample volume is low in order to avoid excessive band-broadening associated with the injection, stipulating an upper limit of 3% of the total capillary volume. (Ref 2-7). As a result, the concentration sensitivity of CE is higher than that typically achieved with LC-MS interfacing. The exceptionally low sample consumption of CE means that analysis of a 1 nL injected zone of a 5 μM solution of a protein with a molecular weight of approximately 20,000 Da results in loading a 0.15 ng mass of protein into the separation capillary. Although the sample mass loading is low, it is compatible with several modes of detection, including mass spectrometry (MS).
The synergy between CE and mass spectrometry has driven interfacing with electrospray ionization (ESI). Many of these CE applications benefit from coupling to MS detection which provides a means to conclusively identify protein structures by the combination of migration time and m/z values. Moreover, MS analyses differentiate protein isoforms, identify post translational modifications, and resolve protein conformations. (Ref 2-8, 2-9). Since the earliest reports of the use of ESI with CE, (Ref. 2-10, 2-11) both sheathless and nanoflow sheath designs (such as e.g., the first example analysis apparatus 100 discussed above in relation to
To date, a handful of CE-ESI-MS analyses of proteins have been performed under native conditions using both sheathless and sheath flow systems. The sheathless interfaces use commercially available instrumentation including capillaries with a modified surface and a porous frit for antibodies (Ref 2-21, 2-22) and large DNA-histone complexes comprising endogenous nucleosomes. (Ref 2-23). The separations are achieved with volatile ammonium acetate electrolytes prepared at, or near, a neutral pH. A conductive liquid, generally comprised of acetic acid, completes the separation circuit without interacting with the protein samples in the separation capillary. The electrophoresis run times for these sheathless systems extend to about 30 minutes. (Ref. 2-21-2-23). The separation is often performed with a superimposed pressure, (Ref 2-21, 2-23) which reduces the separation efficiency (Ref. 2-23, 2-24) because of the band broadening associated with the additional laminar flow. Similar to the sheathless interfaces, the sheath flow CE-ESI-MS analyses of proteins performed under native conditions are based on commercially available instrumentation and separation capillaries that have been modified to eliminate electroosmotic flow. For example, analyses achieved with capillaries modified with a linear polyacrylamide have demonstrated separations of the 70S ribosome complex from E. coli, (Ref. 2-25) proteoforms from E. coli lysate, (Ref. 2-26) and for antibodies. (Ref 2-27). These separations required a superimposed pressure ranging from 0.5 to 1 psi. For the analyses of proteoforms and the 70S ribosome complex, the CE separation times ranged from 6026 to 9025 min. For the focusing experiments the total time for the pre-separation focusing step and separation was 40 min. (Ref. 2-27).
VSSI is an electric field-free alternative to interfacing separations with MS. The interface is operated under ambient conditions and the nebulization process is driven by focusing acoustic energy at a sharp edge. Probe-based liquid interfacing has been coupled to flow rates ranging from 20 to 30 μL/min for the detection of small molecules, (Ref. 2-28-2-32) peptides, (Ref 2-31) DNA (Ref 2-31, 2-33) and proteins. (Ref. 2-31, 2-33-2-35). The geometry and configuration of pulled glass capillary VSSI probes have been adapted to accommodate sub-microliter flow rates inherent in CE. (Ref 2-36, 2-37). The VSSI interfacing has been directly coupled with CE separations in both a sheathless format36 and in a sheath flow design. (Ref 2-37). The sheathless capillary design transfers the analyte bands, generally in the picogram quantities, directly to the MS. This requires a minimum bulk electroosmotic flow of 70 nL/min to sustain the nebulization process. This limitation in the flow rate is overcome when a 400-900 nL/min sheath flow is integrated in the system, allowing the user to develop separations with suppressed and even zero-flow electroosmotic flow. (Ref 2-37). CE separations are achievable for anionic proteins resistant to surface adsorption and in the presence of a bulk electroosmotic flow. (Ref 2-36). These conditions are not applicable to all proteins because the silica surface introduces non-specific adsorption that reduces the separation efficiency or changes the protein structure; for example, through denaturation. These challenges can be overcome by leveraging both CE-VSSI-MS and coating technology, leading to greater flexibility in protein separations with CE-MS technologies.
The techniques discussed herein provide alternative interfacing strategies for protein analyses by CE-MS. The results outlined herein demonstrate that while the ionization process inherent in VSSI is pH independent, it can be important to render the electrophoretic separation pH independent. Moreover, the composition of the sheath fluid can affect the ion suppression and formation of adducts. Proteins of different isoelectric points and molecular mass were evaluated (See e.g., Table S1 shown in
Materials and Methods
Chemicals and reagents. Acetic acid, ammonium acetate (431311), α-chymotrypsinogen A (C4879), cytochrome c (C3131), β-lactoglobulin (L0130), ribonuclease A (R5500), transferrin (T8158), and ubiquitin (U6253) were purchased from Sigma Aldrich (St. Louis, MO). The 4-methylmorpholine (A12158.AP) was purchased from Thermo Fisher Scientific (Waltham, MA). Cetyltrimethylammonium bromide (CTAB) was purchased from Acros Organics (Geel, Belgium). The phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihaxanoyl-sn-glycero-3-phosphocholine (DHPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Deionized water is supplied from an ELGA Purelab and Veolia Chorus I (Lowell, MA).
Sample and solution preparation. Protein powder stocks were dissolved in 50 mM ammonium acetate to the desired stock concentration. The anionic proteins transferrin and β-lactoglobulin and cationic proteins α-chymotrypsinogen A and ribonuclease A were desalted prior to analysis on the unmodified capillary using molecular weight cutoff filters (UFC5010 Millipore Sigma, Allentown, PA). Briefly, the filter was conditioned with 50 mM ammonium acetate. The protein stock was loaded onto the filter and spun down 1-5 times, depending on the protein (14000 g 10 min 19° C.), with 50 mM ammonium acetate. The protein was recovered from the column with an inverted spin (1000 rpm 3 min 19° C.). All proteins were analyzed on a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) to determine the true concentration. Protein stocks were diluted into deionized water for analysis. Preparation of the phospholipid coating has been previously reported.38 The CTAB solution was prepared from powder stock daily using 50 mM ammonium acetate.
Capillary electrophoresis. The electrophoresis capillary has a 25 μm inner diameter and a total length of 30 cm, resulting in a total volume of 150 nL for the separation channel. Capillaries were conditioned prior to use. For bare-fused silica analyses, the capillary is flushed 30 min with 0.1 N ammonium hydroxide, 15 min with water, and 15 min with background electrolyte at 172 kPa (25 psi). Phospholipid coating is applied to the capillary by flushing 3 min with background electrolyte, 20 min with phospholipid, and 3 min with background electrolyte at 172 kPa (25 psi). CTAB coating is applied to the capillary after the phospholipid coating by flushing 7.5 min with background electrolyte, 7.5 min with phospholipid nanogel, 7.5 min with 100 μM CTAB, and 7.5 min with background electrolyte at 172 kPa (25 psi).
VSSI probe fabrication. Solid glass rods (GR100-4 Precision Instruments 1 mm×10 cm) were laser pulled (Sutter Instrument Company, Novato, Ca) under the settings HEAT=475, 650; FIL=4; VEL=60; DEL=130; PUL=50. The pulled probes are trimmed to a final outer diameter of approximately 50 μm. The probe is attached to the bottom of a piezoelectric transducer (7BB-27-4 LO, Murata, diameter=27 mm) using a 5-minute epoxy (14205 Devcon, Danvers, MA). The transducer is then attached to a function generator (bbs Signal Generator, Koolerton, Hong Kong) and amplifier (7500, Krohn-Hite, Brockton, MA). A square wave is applied to each probe and the operating frequency and amplitude range from 80-100 kHz and 0.09-0.10 V peak-to-peak (Vpp). An amplification of 100× provides a Vpp of 9-10 V. The exact frequency and applied voltage used for each probe varies and is established by observing the spray achieved by contacting the probe with a wet cotton swab.
CE-VSSI-MS interfacing and instrumentation. The CE-VSSI-MS nanoflow sheath design has been previously characterized in relation to at least
Results and Discussion
CE-VSSI-MS interfacing. The nanoflow sheath design implemented herein was previously characterized for small molecules as discussed above in relation at least to
Separation of anionic proteins at pH 7.6 in an unmodified capillary. Separations of anionic transferrin (pI 5.7) and β-lactoglobulin variants (pI 5.3) can be accomplished using a background electrolyte buffered to pH of above 7 (e.g., 7.6) and an unmodified fused silica capillary because anions typically do not interact with the negatively charged silica surface. The extracted ion chromatogram in
An advantage of the CE separation (
VSSI-MS sheath composition effect on ion suppression and adduction. CE-ESI-MS incorporating a sheath flow typically uses solutions differing from the separation electrolyte, like acid and organic solvent, to assist in ionization. (Ref 2-46-2-49). As CE-VSSI-MS is a new technique, the effect of the sheath flow composition on analyte response and peak adduction has not been studied previously. The effects of the sheath fluid composition were evaluated with β-lactoglobulin using direct infusion VSSI-MS. This was done to eliminate contributions of analyte desalting provided by the CE separation. The charge state distribution observed in the spectra obtained with direct infusion is comparable to the data obtained with CE. Analyte suppression is reduced in ESI by decreasing sheath fluid ionic strength when using low concentration analytes. (Ref 2-50). This effect is also observed in VSSI as shown in
Cationic proteins require acidic conditions in an unmodified capillary. In contrast to anionic proteins, CE separations of cationic proteins in an unmodified capillary must be designed to address protein adsorption to the negatively charged silica surface. CE-ESI-MS typically utilizes an acidic background electrolyte to reduce this effect, and this strategy has been adapted to CE-VSSI-MS. This is demonstrated in
The CE-VSSI-MS separation of 2.5 μM cytochrome c, 10 μM ribonuclease A, and 10 μM α-chymotrypsinogen A (
Semi-permanent coatings for CE. An alternative to using an acidic background electrolyte to separate cationic proteins is to passivate the capillary surface with a lipid coating to minimize surface adsorption. When the surface charge is masked, the separation can be conducted at physiological pH. The surface of the channel can be covalently modified with polymers that mitigate protein adsorption, (Ref. 2-57, 2-58) and these can be purchased or fabricated by the user. (Ref 2-46, 2-47). Semi-permanent capillary coatings are an alternative to covalently modified capillaries, (Ref. 2-59) which can be modified by simply flushing the capillary. Phospholipid coatings passivate the capillary surface by forming a zwitterionic lipid bilayer on top of the negatively charged silanol groups as depicted conceptually in
Semi-permanent coating for suppressed electroosmotic flow at neutral pH. A CE separation capillary with a semi-permanent lipid coating is achieved with a suppressed electroosmotic flow and a background electrolyte maintained at a pH of 7 for the cationic proteins: cytochrome c, ribonuclease A, and α-chymotrypsinogen A. In contrast to the separation achieved with the unmodified capillary and acidified background electrolyte (
This results demonstrate the successful implementation of a nanoflow sheath CE-VSSI-MS system providing more powerful information for protein analyses. For example, the CE-VSSI MS separations resolved lactoglobulin protein variants and shed light on the degree of glycosylation of anionic transferrin. Moreover, cationic protein separations can be performed using a neutral background electrolyte without excessive surface adsorption while still achieving a rapid separation. Additionally, the intensity of the protein signal is improved by modifying the composition of the low ionic strength sheath fluid to include a serine additive. This novel CE VSSI system enabled detection of proteins at concentrations that are similar to literature reports achieved with CE-ESI systems. In contrast to the ESI interfacing, the VSSI interfacing does not require acidic or organic buffers to enhance ionization. The nanoflow sheath is fully compatible with semi-permanent surface coatings operated under conditions of suppressed (i.e. lipid coating) or sustained (i.e. CTAB-lipid coating) electroosmotic flow, offering flexibility in the separation conditions used to resolve the protein samples. This study demonstrates a proof-of-principle of capillary electrophoresis-VSSI protein separations under physiological pH in the presence of a biocompatible surface coating. Future work will focus on the ability of this design to separate other protein systems, including larger protein complexes and to gain a better understanding about the ionization mechanisms of VSSI for proteins.
VE-VSSI-MS with VSSI Probe having a sheath fluid capillary.
In some example systems, the difference between the flow rates of the analyte liquid in the separation capillary and the sheath fluid in the sheath capillary can result in undesirable dilution of the analyte liquid. For example, if the flow rate of the sheath fluid is considerably greater than the flow rate of the analyte liquid, then the nebulized and ionized spray generated by the VSSI probe can include an undesirably diluted analyte liquid. In one approach, the flow rate of the sheath fluid can be reduced by incorporating the sheath capillary in the VSSI probe instead of over the separation capillary (as shown in
By incorporating the sheath capillary into the VSSI probe, the sheath capillary can be designed independently of the size of the sheath capillary. That is, without having to cover the separation capillary, which can be of different diameters thereby affecting the diameter of the sheath capillary, the sheath capillary incorporated in the VSSI probe can be sized independently of the size of the separation capillary. This helps reducing the cost of the apparatus.
A ground terminal slot 3822 can be positioned at the first end 3808 of the connector body 3806. The ground terminal slot 3822 can extend between an outside surface 3818 of the connector body 3806 and an inner surface 3820 of the conduit 3812. The ground terminal slot 3822 can be configured to removably receive a ground terminal that is part of the CE. The internal diameter or width of the ground terminal slot 3822 can be greater than the corresponding diameter or width of the ground terminal. In some instances, the inner wall of the ground terminal slot 3822 can include an insulating material. The ground terminal can be positioned such that it makes contact with the analyte liquid 108 exiting the separation capillary 104.
The second example connector 3800 can also include an acoustic probe slot 3824 positioned at the first end 3808 of the connector body 3806. The acoustic probe slot 3824 can extend between the outside surface 3818 of the connector body 3806 and an inner surface 3820 of the conduit 3812. The acoustic probe slot 3824 can be configured to removably receive an acoustic probe that has a sheath capillary such as, for example, the VSSI probe 3500 discussed above in relation to
In instances where the connector body 3806 includes the nut portion 3802 and the port portion 3804, the ground terminal slot 3822 and the acoustic probe slot 3824 can be formed in the port portion 3804. In some instances, the ground terminal slot 3822 and the acoustic probe slot 3824 can traverse through both the port portion 3804 and the nut portion 3802 extending between the outside surface 3818 of the connector body 3806 and the inner surface of the conduit 3812. In some instances, the ground terminal slot 3822 or the 3824 or both can be positioned such that the dead stop 3826 is positioned between the slot and the first end 3808 of the connector body 3806.
The connector body 3806 can also include a dead stop 3826 that is formed on an inner surface of the conduit 3812. The dead stop 3826 can have a diameter that is less than the diameter of the separation capillary 104 and can stop the separation capillary 104 from extending out of the conduit 3812 at the first end 3808. In instances where the connector body 3806 includes the nut portion 3802 and the port portion 3804, the conduit 3812 can have a first portion that is formed in the nut portion 3802 and a second portion that is formed in the port portion 3804. The dead stop 3826 can then be formed in the second portion of the conduit 3812 (i.e., in the port portion 3804). In some instances, the dead stop can instead be formed in the nut portion 3802.
The connector body 3806 can be formed of any rigid material such as, for example, metal, thermoplastics or a combination thereof. The connector body 3806 can be built using casting methods, milling methods, CNC methods, 3D printing methods, or other methods know in the art for manufacturing similar types of devices.
Aspects: the following provides details of various aspects in relation to the disclosure.
Aspect 1: This aspect includes an apparatus, including: a separation capillary having an injection end and a distal end, the injection end configured to receive analyte liquid and the distal end configured to expel the analyte liquid; a sheath capillary covering the distal end of the separation capillary, the sheath capillary having a diameter that is greater than an outer diameter of the separation capillary at the distal end, the sheath capillary having a first end and a second end, the distal end of the separation capillary positioned between the first end and the second end of the sheath capillary, the sheath capillary carrying a fluid between the second end and the first end; an acoustic probe configured to vibrate positioned in contact with the at least one of the distal end of the separation capillary or the first end of the sheath capillary; and a ground terminal positioned at the first end of the sheath capillary.
Aspect 2: An aspect in combination with any one of the aspects 1, 3-20, wherein a flow rate of the fluid in the sheath capillary is less than one microliter per minute.
Aspect 3: An aspect in combination with any one of the aspects 1-2, 4-20, wherein a longitudinal axis of the acoustic probe is positioned between 80 degrees to 125 degrees in relation to a longitudinal axis of at least one of the separation capillary or the first end of the sheath capillary.
Aspect 4: An aspect in combination with any one of the aspects 1-3, 5-20, wherein the separation capillary has a length, measured between the injection end and the distal end, between 5 cm and 2 m.
Aspect 5: An aspect in combination with any one of the aspects 1-4, 6-20, wherein the separation capillary has a total volume of no more than 8 micro-liters.
Aspect 6: An aspect in combination with any one of the aspects 1-5, 7-20, wherein an inner diameter of the separation capillary is between 15 micrometers and 35 micrometers.
Aspect 7: An aspect in combination with any one of the aspects 1-6, 8-20, wherein a distance between the distal end of the separation capillary and the ground terminal is less than 2 millimeters.
Aspect 8: An aspect in combination with any one of the aspects 1-7, 9-20, wherein a voltage difference is maintained between the injection end of the separation capillary and the ground terminal.
Aspect 9: An aspect in combination with any one of the aspects 1-8, 10-20, wherein a flow rate of the analyte liquid in the separation capillary is between 0 nano-liters and 70 nano-liters.
Aspect 10: An aspect in combination with any one of the aspects 1-9, 11-20, wherein at least a portion of the inner surface of the separation capillary is coated with lipids.
Aspect 11: An aspect in combination with any one of the aspects 1-10, 12-20, wherein electroosmotic flow within the separation capillary is suppressed.
Aspect 12: An aspect in combination with any one of the aspects 1-11, 13-20, wherein the analyte liquid includes small or large molecules that are positively charged, negatively charged, or neutral charged.
Aspect 13: An aspect in combination with any one of the aspects 1-12, 14-20, wherein the analyte liquid includes cationic proteins and acidic background electrolytes.
Aspect 14: An aspect in combination with any one of the aspects 1-13, 15-20, wherein the analyte liquid includes cationic proteins, and wherein at least a portion of the inner surface of the separation capillary has at least one of a net neutral lipid coating or a hybrid cationic lipid coating.
Aspect 15: An aspect in combination with any one of the aspects 1-14, 16-20, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is semi-permanent.
Aspect 16: An aspect in combination with any one of the aspects 1-15, 17-20, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is positively charged.
Aspect 17: An aspect in combination with any one of the aspects 1-16, 18-20, wherein the analyte liquid includes anionic or neutral proteins and an electrolyte buffer at a pH of above 7.
Aspect 18: An aspect in combination with any one of the aspects 1-17, 19-20, wherein the fluid in the sheath capillary is an electrolyte with neutral pH.
Aspect 19: An aspect in combination with any one of the aspects 1-18, and 20, wherein the fluid in the sheath capillary includes one or more amino acid additives.
Aspect 20: An aspect in combination with any one of the aspects 1-19, wherein the one or more amino acid additives include at least one of L-serine, D-serine, or amino acid additives or L- or D-configuration.
Aspect 21: This aspect includes an apparatus, including: a separation capillary having an injection end and a distal end, the injection end configured to receive analyte liquid and the distal end configured to expel the analyte liquid; an acoustic probe capillary having a probe end and an inlet end, the probe end of the acoustic probe capillary configured to vibrate and positioned in contact with the distal end of the separation capillary, the acoustic probe capillary carrying a fluid from the inlet end to the probe end; and a ground terminal positioned at the distal end of the separation capillary.
Aspect 22: An aspect in combination with any one of the aspects 21 and 23-43, further including: a connector having a conduit for removably receiving the separation capillary, the connector including a slot to accommodate the acoustic probe capillary, wherein the conduit and the slot are positioned such that the probe end of the acoustic probe capillary makes contact with the distal end of the separation capillary.
Aspect 23: An aspect in combination with any one of the aspects 21-22, 24-43, wherein the connector further includes a housing to house a ground terminal, wherein the housing and the conduit are positioned to such that the ground terminal makes contact with the distal end of the separation capillary.
Aspect 24: An aspect in combination with any one of the aspects 21-23, 25-43, wherein a portion of the acoustic probe capillary is coupled with a vibrating structure.
Aspect 25: An aspect in combination with any one of the aspects 21-24, 26-43, wherein the vibrating structure includes a piezoelectric transducer.
Aspect 26: An aspect in combination with any one of the aspects 21-25, 27-43, wherein a flow rate of the fluid in the acoustic probe capillary is less than one microliter per minute.
Aspect 27: An aspect in combination with any one of the aspects 21-26, 28-43, wherein the separation capillary has a length, measured between the injection end and the distal end, between 5 cm and 2 m.
Aspect 28: An aspect in combination with any one of the aspects 21-27, 29-43, wherein the separation capillary has a total volume of no more than 8 micro-liters.
Aspect 29: An aspect in combination with any one of the aspects 21-28, 30-43, wherein a voltage difference is maintained between the injection end of the separation capillary and the ground terminal.
Aspect 30: An aspect in combination with any one of the aspects 21-29, 31-43, wherein an inner diameter of the separation capillary is between 15 micrometers to 35 micrometers.
Aspect 31: An aspect in combination with any one of the aspects 21-30, 32-43, wherein a distance between the distal end of the separation capillary and the ground terminal is less than 2 millimeters.
Aspect 32: An aspect in combination with any one of the aspects 21-31 33-43, wherein a flow rate of the analyte liquid in the separation capillary is between 0 nano-liters and 70 nano-liters.
Aspect 33: An aspect in combination with any one of the aspects 21-32, 34-43, wherein at least a portion of the inner surface of the separation capillary is coated with lipids.
Aspect 34: An aspect in combination with any one of the aspects 21-33, 35-43, wherein electroosmotic flow within the separation capillary is suppressed.
Aspect 35: An aspect in combination with any one of the aspects 21-34, 36-43, wherein the analyte liquid includes small or large molecules that are positively charged, negatively charged, or neutral charged.
Aspect 36: An aspect in combination with any one of the aspects 21-35, 37-43, wherein the analyte liquid includes cationic proteins and acidic background electrolytes.
Aspect 37: An aspect in combination with any one of the aspects 21-36, 38-43, wherein the analyte liquid includes cationic proteins, and wherein at least a portion of the inner surface of the separation capillary has at least one of a net neutral lipid coating or a hybrid cationic lipid coating.
Aspect 38: An aspect in combination with any one of the aspects 21-37, 39-43, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is semi-permanent.
Aspect 39: An aspect in combination with any one of the aspects 21-38, 40-43, wherein the at least one of a net neutral lipid coating or a hybrid cationic lipid coating is positively charged.
Aspect 40: An aspect in combination with any one of the aspects 21-39, 41-43, wherein the analyte liquid includes anionic or neutral proteins and an electrolyte buffer at a pH of above 7.
Aspect 41: An aspect in combination with any one of the aspects 21-40, 42-43, wherein the fluid in the acoustic probe capillary is an electrolyte with neutral pH.
Aspect 42: An aspect in combination with any one of the aspects 21-41 and 43, wherein the fluid in the acoustic probe capillary includes one or more amino acid additives.
Aspect 43: An aspect in combination with any one of the aspects 21-42, wherein the one or more amino acid additives include at least one of L-serine, D-serine, or amino acid additives or L- or D-configuration.
Aspect 44: This aspect includes a connector, including: a connector body having a first end and a second end; a conduit extending between the first end and the second end of the connector body, the conduit configured to removably receive a separation capillary, wherein a portion of the conduit at the first end of the connector body accommodates a distal end of the separation capillary; and an acoustic probe slot positioned at the first end of the connector body, the acoustic probe slot extends between an outside surface of the connector body and the inner surface of the conduit, the acoustic probe slot configured to removably receive an acoustic probe having a sheath capillary.
Aspect 45: An aspect in combination with any one of the aspects 45-51, further including: a ground terminal slot positioned at the first end of the connector body, the ground terminal slot extends between an outside surface of the connector body and an inner surface of the conduit, ground terminal slot configured to removably receive a ground terminal.
Aspect 46: An aspect in combination with any one of the aspects 44-45, 47-51, further including: A dead stop positioned in the conduit at the first end of the connector body, the dead stop configured to have a diameter that is less than a diameter of the separation capillary such that the separation capillary is prevented from being pushed out of the conduit at the first end.
Aspect 47: An aspect in combination with any one of the aspects 44-46, 48-51, wherein the connector body is formed of at least one of metal or thermoplastic.
Aspect 48: An aspect in combination with any one of the aspects 44-47, 49-51, wherein the connector body includes a dead stop formed on an inner surface of the conduit, wherein the dead stop has a diameter that is less than the diameter of the separation capillary.
Aspect 49: An aspect in combination with any one of the aspects 44-48, 49-51, wherein the connector body includes: a nut portion and a port portion, wherein the nut portion screws into the port portion, wherein a portion of the conduit is formed in the nut portion and a second portion of the conduit is formed in the port portion, and a dead stop formed on the inner surface of the second portion of the conduit in the port portion, wherein the dead stop has a diameter that is less than the diameter of the separation capillary.
Aspect 50: An aspect in combination with any one of the aspects 44-49 and 51, further including; a ground terminal slot positioned at the first end of the connector body, the ground terminal slot extends between an outside surface of the connector body and an inner surface of the conduit, ground terminal slot configured to removably receive a ground terminal, wherein the ground terminal slot is formed in the port portion.
Aspect 51: An aspect in combination with any one of the aspects 44-50, wherein the acoustic probe slot is formed in the port portion.
References: All cited references, patent or literature, are incorporated by reference in their entirety. The examples disclosed herein are illustrative and not limiting in nature. Details disclosed with respect to the methods described herein included in one example or embodiment may be applied to other examples and embodiments. Any aspect of the present disclosure that has been described herein may be disclaimed, i.e., exclude from the claimed subject matter whether by proviso or otherwise.
Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other implementations without departing from the spirit or scope of this disclosure. Thus, the claims are not intended to be limited to the implementations shown herein, but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein.
This application claims priority to U.S. Provisional Application No. 63/393,816, filed Jul. 29, 2022, entitled “Devices and Processes for Mass Spectrometry Utilizing Vibrating Sharp-edge Spray Ionization,” incorporated by reference herein in its entirety.
This invention was made with government support P41 GM128577 awarded by the National Institutes of Health; and CHE2004021 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63393816 | Jul 2022 | US |
