SE2130189A1 - Apparatus and method for capillary isoelectric focusing of biomolecules - Google Patents

Abstract

In capillary isoelectric focusing (CIEF) of molecules, the resolving power positively correlates with the physical length of the separation capillary. But increasing the length of the separation capillary results also in an increase in the separation time since each analyte in a longer capillary must migrate on average a greater distance to its equilibrium point. The present disclosure provides a method for improved CIEF resolution without significant increase in focusing time by using at least two isoelectric focusing capillary sections of similar volume, but different diameters and linear lengths, with each subsequent focusing capillary sections being longer than the previous one.

Description

TITLE Apparatus and method for capillary isoelectric focusing of biomolecules FIELD OF THE DISCLOSURE The present disclosure is related to the field of biomolecule separation, particular by capillary isoelectric focusing. The biomolecules could come from biological samples, such as bodily fluids, tissues, cells or microorganisms, recombinantly expressed proteins, biosimilars or food stuff. BACKGROUND OF THE DISCLOSURE Capillary isoelectric focusing (IEF) is an electrophoretic technique to separate amphoteric compounds, such as proteins and peptides as well as other biomolecules, from a complex mixture. Every compound has isoelectric point (pl) which corresponds to pH at which the molecule becomes electrically neutral. ln CIEF separation, the separating capillary is filled with slightly conducting buffer containing analyte molecules as well as ampholytes, which are amphoteric molecules that contain both acidic and basic groups. When electric field is set up along the capillary, the ampholyte molecules move along it, establishing the pH gradient. The analyte molecules also move along the capillary until they reach the pH point equal to their pl. The resolving power of CIEF depends upon the capillary length, with greater lengths providing higher resolution. But as initially the analyte molecules are homogeneously distributed along the capillary, the time it takes them to focus into the point with pH=pl increases significantly with the length of the capillary. The focusing time is one of major factors limiting the resolving power, and thus the usefulness, of CIEF as analytical technique.

SUMMARY OF THE DISCLOSURE The present disclosure provides a method for improved CIEF resolution without significant increase in focusing time by using at least two isoelectric focusing sections of similar volume, but different diameters and linear lengths. The sections are connected in tandem, with each subsequent section having longer length than the previous one.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic drawing illustrating tandem capillary isolectric focusing device.

Figure 2. Full width at half maximum (FWHM) of the elution time of a biomolecule versus its focusing time in tandem CIEF device versus one-stage devices (Only L1 and Only L2).

DETAILED DESCRIPTION OF THE INVENTION CIEF can be used to separate polypeptides before analysis by a combination of liquid chromatography and mass spectrometry (LC-MS). As an example, we have previously shown that that such CIEF fractionation increases the depth of the proteome analysis (Pirmoradian, M.; Zhang, B.; Chingin, K.; Astorga-Wells, J.; Zubarev, R. A. Membrane- assisted isoelectric focusing device as a micro-preparative fractionator for two dimensional shotgun proteomics, Anal. Chem, 2014, 86, 5728-5732). ln order to achieve high resolution in CIEF, one needs to employ a long capillary. However, long capillaries are associated with long focusing times. Because initially the analyte molecules more or less homogeneously distributed along the capillary, doubling the capillary length should roughly double the focusing time. Here we provide a tandem design of CIEF that increases the resolving power without significant increase of the focusing time.

The core of the invention is the use of two or more fractionation capillaries connected in tandem. The first capillary is short and serves for preliminary fractionation of the molecules, while the second longer capillary is used for post-focusing of the preliminary fractionated molecules. lmportantly, the volumes of both capillaries are similar, and thus an analyte volume occupies the same fraction of both capillaries.

The principle of invention is explained in Figure 1. ln the following example, we provide some numerical values for the sake of clarity, but in different implementations of the invention these values can be different.

The sample containing analytes and amphylites is first injected in the short (L1 = 3 cm) first capillary connected to the volumes with buffers of different pH values, as in the multijunction CIEF (Pirmoradian, M.; Astorga-Wells, J.; Zubarev, R. A. Multijunction Capil/ary lsoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables lsoelectric Point Fractionation of lntact Human Plasma Proteins for Biomarker Discovery. Anal Chem. 2015, 87, 11840-6). When the voltage 1 is applied along the capillary, the analyte moiecules quickly fractionate, each occupying a region with AL1=0.1 cm around the position with pH=pl. After that the content of the first capillary is moved to the second capillary. During the movement (mobilization), the focused peaks of individual types of biomolecules undergo diffusion, roughly doubling in volume. The second capillary is 15 cm, i.e. 5 times longer than the first capillary, while their volumes are the same. Therefore, after mobilization each type of analyte moiecules will occupy 2 * 5 = 10 times longer volume in capillary 2 than in capillary 1 after focusing, i.e. 1.0 cm instead of 0.1 cm. Yet this zone is much smaller than the full length of capillary 2, which the moiecules would occupy if fractionation only occurred in capillary 2. Thus the focusing time in the second stage of the tandem CIEF device is much shorter than in the same-length capillary of the single-stage CIEF device. And despite the fact that some time was spent in the tandem CIEF device on pre-fractionation in the first section, the overall focusing time in the tandem device should be shorter than in the single-stage device at the same resolving power. Alternatively, higher resolving power should be possible to achieve with the tandem device than with the conventional single- stage device at the same fractionation time.

IMPLEMENTATION The concept of tandem CIEF was verified using the set-up combining in tandem two sections, each created according to literature (Chingin, K.; Astorga-Wells, J.; Pirmoradian Najafabadi, M.; Lavold, T.; Zubarev, R. A. Separation of polypeptides by isoelectric point focusing in electrospray-friendly solution using multiple-junction capillary fractionator, Anal. Chem. 2012, 84, 6856-6862). The first focusing section was 5 cm long, and the second section was 20 cm ling but had nearly the same volume. The two sections were connected by a 4 cm long capillary.

Concentrated aqueous solution of two standard proteins were prepared with following concentration: myoglobin - 13.6 mg/mL, cytochrome C - 6.6 mg/mL, and kept in refrigerator at 5° C. Fresh urea stock solution with 13.3 mg/mL concentration was prepared anew every time. The carrier buffer with ampholytes consisted of 6% Pharmalyte (pH 6-9), 30% glycerol (v/v), and 5% 2-propanol (v/v). Samples were added to the carrier buffer, centrifuged at 10,000 rpm for 5 min, and the supernatant was collected by syringe.

For electrolyte solutions (pH=3 and pH=10), the stock solution of 10% formic acid and 0.5M ammonia was prepared and kept at room temperature. The stock solution was used to prepare fresh: pH=10 buffer - 0.5 mL of 0.5 M ammonia in 25 mL Milli-Q water, and pH=3 buffer - 35 pL of 10% formic acid in 25 mL Milli-Q water. These buffers were stored at room temperature.

A syringe pump (Harvard apparatus, model 55-2111) supplied the carrier buffer flow during the experiment. A VICI Valco Two Position Microelectric Valve Actuator (Model EHMA with 6 Port Valve, USA) with a 5 uL injection volume was used to inject the sample into the CIEF device. A power supply (Bertan, model ARB 30, USA) was used to provide voltage and electrical current through the capillaries. The capillary was obtained from VICI (Scantec Nordic; Sweden). The capillary material was Fluorinated Ethylene Propylene (FEP): 1/32" OD x 0.380 mm ID for the first stage and 1/32" OD X 0.20 mm ID for the second stage. For the intermediate and exit capillaries, PolyEther Ether Ketone (PEEK) was used - 1/32" OD X 0.180 mm ID and 0.100 mm ID, respectively. Tubular Nafion membranes (OD 610 pm, ID 330 pm) were connecting the capillaries to provide electrical contact with platinum electrodes inside the 1.5 mL Eppendorf tubes containing buffers.

For each experiment with a fixed focusing time, half of that time was used in the tandem device for pre-fractionation in the first section followed by elution into the second section, and the remaining time was used for fractionation in the second stage. ln the single- stage experiments, all time was used for focusing.

After focusing, the analyte mixture was eluted and collected into a 384-well plate, with the total elution time of 24 min, and 24 one-minute fractions collected. These fractions were then analyzed off-line by UV-Vis measurements using Epoch Microplate Spectrophotometer (USA). The protein peak width was determined as the average full width at half maximum (FWHM) for two proteins.

The results of the proof-of-principle experiment can be seen in Figure 2. At 50 min focusing time, the tandem CIEF device reached FWHM of5 min, while single-stage CIEF gave FWHM of 7 min and 9 min for focusing in L1 and L2, respectively. To reach FWHM of 5 min, it took the L2-based single-stage CIEF device =9O min, i.e. almost double as much as with the tandem device.

Claims (2)

1. A method for performing separation of biomolecules by capillary isoelectric focusing comprising: (a) The use of at least two connected in tandem isoelectric focusing sections of essentially same volume, wherein each preceding section has a wider inner diameter and shorter linear length than the subsequent section; (b) separation in the preceding section of a mixture of biomolecules with different isoelectric points by isoelectric focusing, followed by; (c) transfer the mixture separated in a preceding section to a subsequent section; (d) isoelectric focusing in the subsequent section.

2. The method according to claim 1, wherein the separated molecules are detected online or immobilized for detection off-line by any other available analytical method, including but not limited to UV-absorbance, fluorescence, and mass spectrometry.