Protein Sample Ultrafiltration Protocols

Amicon® Ultra centrifugal filters are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free and bound species. One of the most common applications for Amicon® devices is concentration and desalting of column fractions during protein purification by various chromatography methods.

Use this protocol for:

Amicon® Ultra Method for Concentration, Desalting or Buffer Exchange

NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.

Principles

During the first centrifugation step, the concentration of macromolecule solute increases; however, filtration removes lower MW solutes, thereby maintaining the salt concentration at 500 mM. For the second spin, another volume of salt-free buffer or water is passed over the retentate, and salt is removed by filtration. This process of “diafiltration” can be repeated to achieve maximum salt removal, or complete buffer exchange.

Amicon® Ultra centrifugal devices allow >90% salt removal in the first centrifugation step. As seen in the table below, one additional centrifugation step generally increases the salt removal to 99% with >90% recovery of the sample.

	Cytochrome c (0.25 mg/mL)		BSA 1 mg/mL		BSA 1 mg/mL		IgG 1 mg/mL
Device	Amicon® Ultra 15 mL filter with 10 kDa NMWL		Amicon® Ultra 15 mL filter with 30 kDa NMWL		Amicon® Ultra 15 mL filter with 50 kDa NMWL		Amicon® Ultra 15 mL filter with 100 kDa NMWL
Spin	1	2	1	2	1	2	1	2
% Protein Recovery	97.3	95.6	96.0	94.4	98.9	92.4	99.9	97.1
% NaCl Removal	97.9	99.9	98.2	99.9	97.1	99.9	97.7	99.5

Table: Removal of sodium chloride and recovery of protein with Amicon® Ultra-15 mL devices.

Three Amicon® Ultra-15 devices of each NMWL indicated were tested with 15 mL of each protein solution containing 500 mM initial concentration of NaCl. Each spin was performed at 4,000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q® water system. NaCl removal was calculated by assaying each sample for conductivity (µS/cm) using a standard conductivity meter (Innolab® Conductivity Meter). Protein recovery was calculated by measuring the final amount of protein in the retentate after each spin and comparing to the amount of protein loaded. Amount of protein in the retentate was calculated by measuring the absorbance of the retentate solution at 410 nm (for Cytochrome c) and at 280 nm (for BSA and IgG) and recovery calculated using the following equation:

Where ...

W_c = total weight of retentate before assay

W₀ = weight of original starting material

C_c = concentration of protein in retentate

C₀ = original starting material concentration

NOTE: Factors independent of the ultrafiltration device may contribute to low protein recovery. Two examples:

• Desalting below a certain threshold for a given protein can result in protein precipitation and lower measured protein recovery.
• Overconcentration of protein can result in lower measured protein recovery.

For single-spin buffer exchange of small samples and with simultaneous protein concentration, refer to the Amicon® Pro system protocol for continuous diafiltration and concentration.

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Even more efficient desalting with simultaneous macrosolute concentration can be achieved using the Amicon® Pro device, which was specifically engineered for effective buffer exchange (≥ 99%) with a single spin (see figure below). Furthermore, the Amicon® Pro device enables continuous diafiltration. Continuous diafiltration maintains a constant volume throughout the buffer exchange process, by matching the rate at which exchange buffer is introduced to the filtrate flow. Because the volume remains constant, the concentration of retained solute also remains constant, providing a gentler method of buffer exchange.

As shown in another protocol, buffer exchange is thus completed in a single step, instead of three steps as required for traditional diafiltration methods (see data comparing traditional and continuous diafiltration).

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Amicon® Pro Method for Desalting and Concentration:

*For larger sample/buffer volumes, spin time will need to be increased appropriately. Please consult user guide for specific instructions.

NOTE: Do not load more than 2 mg total protein in step 5. Exceeding 2 mg will result in protein precipitation upon concentration.

Results

With continuous diafiltration, as occurs in the Amicon® Pro device, the sample (after it has undergone initial concentration and has entered the Amicon® Ultra-0.5 mL component of the Amicon® Pro system) remains at a constant volume (and concentration) until the entire quantity loaded in the upper reservoir has been passed through the exchange device tip. In this experiment, over 99.9% of the salt was removed after a single spin (See table below). The Amicon® Pro device also afforded extremely high protein recovery, having, on average, < 2% loss of starting BSA.  

	BSA 0.05 mg/mL
Device	Amicon® Pro device (with Amicon® Ultra 10 kDa NMWL filter attached)
Spin	% Protein Recovery	% NaCl Removal
1	Not determined (initial protein concentration step)	0% (NaCl concentration unchanged during proteinconcentration step)
2	>98%	>99.9%

Table: Removal of sodium chloride and recovery of protein with Amicon® Pro device.

500 µL of a BSA solution was subjected to simultaneous desalting (using 1 M Tris, pH 7.5) and concentration. The resulting sample was brought to an equivalent volume in 1 M Tris pH 7.5, diluted 1:50 in Milli-Q® H20, and assayed for conductivity (µS/cm) using a standard conductivity meter (InoLab® Cond). Protein concentration was determined using the Direct Detect® Infrared Spectrometer for Protein Quantitation.

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Detergents are frequently used to solubilize proteins and nucleic acids during purification, but the presence of detergents may interfere with downstream analyses. Free detergent can be removed by various protocols¹; however, detergent molecules that are noncovalently bound to macromolecules can be displaced but not completely removed. Fortunately, for most downstream analyses, it is sufficient to remove the free detergent.

Amicon® Pro centrifugal filters are efficient tools for removing detergents from solutions of proteins or nucleic acids. The choice of filter depends on the critical micelle concentration (CMC) for a given detergent. At concentrations above the CMC, detergent monomers form micelle aggregates with gross changes in molecular structure. Choosing a centrifugal device with the correct nominal molecular weight limit (NMWL) allows for effective detergent removal.

However, it may be challenging to choose an ultrafiltration device with a NMWL that allows detergents, but not protein of interest, to pass through. Because they form large micelles, many detergents require using membranes with NMWL as high as 100 kDa for size-based depletion, resulting in loss of the proteins of interest in the sample. One way to use smaller pore size ultrafilters for these detergents is to reduce micelle formation first, by sample dilution.

The Amicon® Pro system was used to remove detergents using continuous diafiltration, in which a constant sample volume is continously washed with detergent-free buffer. This method could remove detergents more efficiently and with higher protein recovery than using discontinuous diafiltration or dialysis. Simultaneous monitoring detergent removal and protein recovery was performed using the Direct Detect® infrared spectrometer.

For more information on detergent removal monitoring methods, please refer to our publication, Detergent Analysis in Protein Samples Using Mid-Infrared (MIR) Spectroscopy.

Method

Example Results of Detergent Removal Using the Amicon® Pro System

Removal of 0.5% sodium deoxycholate from 2 mg/mL IgG. Using 2 mg/mL IgG solubilized in 0.5% sodium deoxycholate, the Amicon® Pro purification system was used to show that detergent diluted below its CMC could be easily removed by a size-based ultrafiltration method.

The figure below compares detergent removal via continuous diafiltration with successive dilution and concentration steps. Continuous diafiltration keeps the concentration of free detergent below the critical micelle concentration (CMC). As a result, fewer micelles form than with alternating rounds of dilution and concentration, and ultrafiltration is more effective at removing detergent molecules.

Continuous diafiltration also avoids concentrating protein too rapidly in a detergent-free buffer, a process which may disrupt stabilizing detergent-protein interactions, cause protein precipitation, and reduce yield.

Measuring detergent removal and protein recovery during detergent removal made it possible to identify the protocol that yielded the optimal balance of both processes.

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Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution. Stirred cells are gentle, efficient, and feature a removable membrane for greatest flexibility. However, centrifugal ultrafilters have their own advantages:

• Devices are pre-assembled and ready to use
• Can process 15 – 65 mL volumes; use multiple devices for larger volumes 
• Spin times are fast (minutes)
• No need to refill the reservoir (less contamination risk)

The following protocol is an example that demonstrates the use of Centricon® Plus-70 centrifugal filter devices to purify and concentrate a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose® (GE) beads to produce an affinity column for isolating human IgE antibodies. (Note: protocol courtesy of GKT School of Biomedical Sciences, London.)

Method

Results

According to spectrophotometric analysis, 18.8 mg/L of alpha-gamma fusion protein was recovered using the Centricon® Plus-70 devices (data not shown). The HPLC trace analysis revealed the profile of the alpha gamma fusion component of the human Fc_εRI (See figure below).

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Affinity interaction chromatography is an effective method for protein purification that can achieve up to 95% purity in one step. It is often employed in the small-scale batch mode as a quick method for microgram-scale protein purification. The typical protocol involves: Pre-packed mini-spin columns (such as Montage® Antibody Purification Kits) for protein purification simplify this process into a one-hour protocol.

For a more flexible protocol, centrifugal devices with a microporous membrane, such as Ultrafree®-MC centrifugal devices, hold the sample and affinity resin in a filter basket, thus creating a “home-made” mini-spin column. These devices combine the high efficiency of batch binding and washing with the handling convenience of a mini-spin column.

With minimal hands-on time, the method provides flexibility in resin-to-lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. Here, the method was used to purify rabbit IgG on ProSep® resin and His-tagged C-RP protein on three different commercial metal-chelate resins.

Method for IgG Purification

Solutions

• ProSep-A binding buffer A: 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0
• ProSep-A elution buffer B2: 0.2 M Glycine/HCl, pH 2.5
• ProSep-A neutralization buffer: 1 M Tris/HCl, pH 9.0

Procedure

Method for His-tagged C-RP Purification using Ultrafree® devices

Solutions

• Lysis buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
• Binding buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
• Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM immidazole, pH 7
• Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 250 mM immidazole, pH 7
• 1 mg/mL Lysozyme stock
• Benzonase

Procedure

Results

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Traditional centrifugation-based affinity purification presents certain challenges. Batch processing of resins directly in microcentrifuge tubes is subject to sample loss during aspiration. Spin columns alleviate this concern; however, due to their limited volume capacity (typically 0.5 mL), the protocol is tedious, requiring multiple spin cycles during the wash and elution steps. Moreover, while most purification workflows require additional sample neutralization, buffer exchange, and/or concentration prior to downstream analysis, no single traditional centrifugation device is capable of performing all steps in the purification process.

The Amicon® Pro purification system is an adaptable centrifugal device that couples affinity-based spin column purification with downstream sample concentration and buffer exchange. By condensing the protein preparation workflow, the Amicon® Pro system eliminates the need for multiple sample transfers, thereby minimizing protein loss.

Amicon® Pro Protocol for Affinity Purification

This protocol was derived from the study described in the application note, The Amicon® Pro purification system: condensing the protein purification workflow into one centrifugal device. The method was designed for the affinity purification of GST- or His-tagged recombinant protein (from 0.5 mL of lysate).

NOTE: For all protocols using the Amicon® Pro device, all steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.

Add 100 µL Resin (50% Ni-NTA agarose or glutathione agarose slurry) and 1.5 mL Wash buffer to exchange reservoir. Spin 1000 x g for 1 min.

Results

33% faster, less hands-on time. The Amicon® Pro device offered a significant reduction in overall processing time (33%, a savings of 63 min) while also minimizing hands-on time by decreasing the spin steps (3 to 1, in each case) required during the wash, elution, concentration, and buffer exchange phases. SDS-PAGE showed that, despite fewer washes in the Amicon® Pro protocol, high yield and purity were maintained (See figure below).





Elution without dilution. For the elution phase, a single one-minute spin was sufficient to release >90% of bound protein (See figure below). Moreover, when the included Amicon® Ultra 0.5 mL filter was attached, the sample underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin. By combining these two steps, the Amicon® Pro device further eliminated the tedious requirement of determining protein content across the various eluted fractions before concentrating.

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Commercially available protein labeling kits provide researchers with the flexibility to customize their own immunoassay detection panels. However, because many protocols require buffer exchange prior to and following antibody labeling, dialysis-based workflows are time-consuming and subject to significant protein loss at multiple points of sample transfer.

For removing unincorporated label in protein labeling applications, centrifugal devices are a fast, convenient, high-recovery alternative to gel filtration.

Small-Scale (25 μg – 200 μg) Antibody Labeling Using the Amicon® Pro Centrifugal Filter

Significantly higher yields can be achieved from antibody labeling by combining the labeling step with the clean-up step in the all-in-one Amicon® Pro system. The Amicon® Pro purification system is an adaptable centrifugal device coupling affinity purification with downstream sample concentration and buffer exchange.

Two attributes of the Amicon® Pro device make it a convenient device for preparing pure, labeled antibody: For details on the Amicon® Pro Antibody Labeling application, please visit our dedicated Antibody Labeling page, as well as our publication (Read our 2015 paper in Journal of Immunological Methods)

Read below for a brief summary of the protocol for using the Amicon® Pro device for small–scale antibody labeling.

NOTE: All steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.

Optional: Pre-wet device using 0.5 mL TBS-T. Spin at 1000 x g for 1 min.

Sample Results of Antibody Labeling Protocol

	Traditional 0.5 mL centrifugal  diafiltration device	Amicon® Pro Purification System
Sample clean-up	45 min	15 min
Biotinylation reaction	120 min	30 min
Remove free biotin, buffer exchange, concentrate	45 min	15 min
Total protocol time	3.5 hours	1 hour
Antibody recovery	40%	72%

Table: Comparison of biotinylation workflows with respect to time and antibody recovery.

To compare the functional performance of antibodies labeled using traditional centrifugal diafiltration and using the Amicon® Pro method, their detection ability was compared with that of a species/anti-species GAPDH detection pair. Using similar amounts of primary antibody for each blot, both biotin/SA pairs demonstrated the same, if not slightly better sensitivity, than the species/anti-species detection pair (See figure below).

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Using a standard Amicon® Ultra centrifugal filter, a few rounds of diafiltration can efficiently remove unincorporated label from an antibody labeling reaction without diluting the sample. Use the protocol below to remove unreacted fluorescent labels using centrifugal ultrafiltration devices.

Removal of unreacted fluorescent label from antibody conjugation reaction

Results

Figure 1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is visible in the starting material and in the first filtrate. The majority of the free label is removed following one ultrafiltration cycle; free FITC is virtually undetectable after subsequent filtration cycles. Fluorescence measurement data in Figure 2 confirm this result. The free FITC signal is reduced by ~ 80% after the first ultrafiltration, and further reduced to 98% removal after subsequent filtration cycles. This demonstrates the viability of ultrafiltration as an alternative to gel filtration for clean-up of protein labeling reactions.

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Introduction

Monoclonal antibodies (MAb) continue to gain importance as therapeutic and diagnostic agents for many cancers. The process of screening hybridoma libraries for candidate MAbs is both time consuming and labor intensive. Once a hybridoma cell line expressing the desired MAb is established, a bench-scale purification methodology (e.g., 50 to 500 mL) must be developed to produce sufficient MAb for further characterization. A traditional method for purifying MAbs involves these steps: This process typically requires several days to complete and can be labor-intensive when evaluating multiple MAbs in parallel. Ultrafiltration-based MAb purification, on the other hand, is easier, faster (2–3 hours versus 2 days), and yields higher recoveries.

Method

The method involves clarification of the hybridoma supernatant by microfiltration using a Stericup vacuum filter cup, followed by concentration using ultrafiltration¹ with a Centricon® Plus-70 device:

Results

Ultrafiltration was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E10² (See figure 1 below). The purified MAb performed comparably to traditionally/commercially prepared MAbs in Western blotting and ELISA assays (See figure 2 below). Our data show that the combination of microfiltration and ultrafiltration is a rapid method for Mab purification. Additionally, MAb purified on Montage® centrifugal columns with Protein G media is comparable to the commercially available MAb in purity, activity and cost.

Figure 1	Figure 2
Figure 1. Purification of anti c-myc antibody using ultrafiltration. C: clarified supernatant; Tr: Supernatant precipitated with ammonium sulfate and dialyzed using 10K MWCO membrane (Spectrapor, Rancho Dominguez, CA, USA); UF: Supernatant concentrated by ultrafiltration on Centricon®-Plus 70 device. Figure 2. Upper panel shows activity comparison measured in a Western blot between Mab (derived from HEK 293 cells) purified using ultrafiltration versus traditional methods. Three-fold serial dilutions of HEK 293 cell nuclear extracts were run on 4-12% SDS-PAGE gels and transferred to Immobilon®-P membrane. Blots were probed with the indicated primary antibodies and secondary anti-mouse alkaline phosphatase conjugate (MilliporeSigma). The antibodies were detected with alkaline phosphatase substrate and imaged on a Kodak imager. Bottom panel shows activity comparison measured in an ELISA. HEK 293 cells were grown on 96-well plates. After fixation and epitope retrieval by heating for 10 minutes in a microwave oven, cells were permeabilized with 1% saponin (MilliporeSigma) in PBS and 2% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions of the indicated purified MAbs. The cells were then washed and treated with goat anti-mouse HRP conjugate antibody (MilliporeSigma). The reactions were developed using a SureBlue™ TMB HRP substrate (KPL, Gaithersburg, MD, USA). The readings were measured on a SpectraMax® plate reader (Molecular Devices, Sunnyvale, CA, USA).

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If an antibody must be purified from a small sample volume (0.5-10 mL), then the Amicon® Pro Purification System is recommended. Because this centrifugal device enables purification, buffer exchange, and concentration to be performed without transferring the sample or diluting it, the risk of antibody loss is minimized. The following method was developed for affinity purification of antibodies (0.5 mL sample) using protein A/G agarose resins in the Amicon® Pro System.

Method

Results

IgGs from rabbit serum (Cat. No. S20-100ML were purified on Protein A or Protein G agarose, using either the Amicon® Pro System (10kDa MWCO, Cat Nos. ACK5010PA and ACK5010PG) or traditional spin columns. Purified IgGs were then buffer-exchanged and concentrated, either in the same Amicon® Pro devices or transferred from spin columns to separate Amicon® Ultra devices.

When the Amicon® Pro System was used, the process required 6 pipetting steps/spins, as compared to 11 pipetting steps/spins required for traditional spin columns and concentrators. As shown in the figure below, antibody recovery remained unchanged. 

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Introduction

Purification protocols for immunoglobulins generally include affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. The resulting purified antibodies are often too dilute for downstream studies or long-term storage. Dialysis is often necessary to concentrate the antibody and/or exchange the buffer into one that preserves protein activity. Ultrafiltration achieves the same result faster, with up to 99% immunoglobulin recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange protocol).

Method and Results

Purification and concentration of Rabbit IgG with Prosep® A and Prosep® G Montage® Antibody Purification Kits and Amicon® Ultra-15 mL centrifugal filters.

A. Purification of IgG using Montage® Kits B. Concentration of IgG using Amicon® Ultra-15 30kDa MWCO devices.

Figure 1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A 20-minute centrifugation step resulted in >95% recovery of immunoglobulins. Figure 2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration. The data demonstrate the suitability of Amicon® Ultra devices for concentrating purified IgG.

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The human plasma proteome is a vast resource for biomarker discovery and investigation. Traditionally, gel filtration chromatography has been used to fractionate protein solutions based on size^1,2. However, gel filtration is laborious, limited by sample size, and time-consuming. It also dilutes the sample significantly. These limitations can be overcome by ultrafiltration, which has been reported^3–5 as a sample preparation method for preparing low molecular weight (<10 kDa) fractions for biomarker analysis. Here, Amicon® Ultra devices were used to purify cytochrome c from a mixture containing 10% fetal bovine serum.

Method

In this experiment, ultrafiltration devices were used to enrich both low molecular weight (LMW) and high molecular weight (HMW) fractions from serum. Using a serial filtration approach (Figure 1), proteins were fractionated through decreasing molecular weight cutoff (MWCO) in Amicon® Ultra 4 mL devices ranging from 100 kDA to 10 kDa MWCO.

Results

 

This serial enrichment strategy enabled protein compartmentalization and improved throughput as compared to direct filtration using a lower MWCO device (Figure 2, above). Samples prepared using the enrichment stragegy also showed improved purity compared to traditional direct filtration (Figure 3, above). The data show that serial ultrafiltration is a viable approach for size-based protein enrichment.

References

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Accurate measurement of specific proteins in urine is important for diagnosing and managing diseases. However, the concentration of urine proteins is often below the detection limit of clinical tests. Amicon® Ultra-4 mL 10 kDa MWCO devices can be used to concentrate urine samples prior to clinical laboratory analyses.

Method

Enrichment of free immunoglobulin light chains (Bence-Jones proteins) in urine samples using Amicon® Ultra-4 mL 10 kDa MWCO filters.

Additional Notes:

Related Products:

Acknowledgements

Research using Amicon® Ultra devices for urine concentration in this protocol was conducted by Mark Merchant, Ph.D. at Helena Laboratories, Beaumont, TX.

References

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Two-dimensional electrophoresis (2DE) is one of the most commonly used methods in proteome analysis. Briefly, proteins are separated by their isoelectric point (first dimension separation) followed by SDS-PAGE separation by molecular weight (second dimension separation). The salts and ionic detergents routinely used in sample preparation are not compatible with 2DE; the combination of high salt concentration and low protein content interferes with isoelectric focusing. One way to concentrate proteins is by acetone or TCA precipitation. However, this method has two key disadvantages:

• Some proteins become insoluble and cannot be resolubilized in IPG buffer
• Many salts become insoluble in acetone and precipitate along with the proteins

Method

Forty grams of bovine liver were blended in an industrial blender in 50 mL 50 mM Tris-HCl, 150 mM NaCl, containing protease inhibitor cocktail. The lysate was cleared by centrifugation, aliquoted, and stored at -20 °C. Protein concentration of 5 mg/μL was determined by BCA protein assay using bovine serum albumin standard.

For 2-DE analysis of the neat lysate, 150 μL of the extract was mixed with 50 μL IPG rehydration buffer and used to rehydrate an IPG strip. For 2-DE analysis of lyophilized lysate, the dried sample was resolubilized in 200 μL of IPG rehydration buffer, cleared by centrifugation, and used to rehydrate an IPG strip.

For 2-DE analysis of acetone-precipitated proteins, 150 μL liver lysate was mixed with 4 volumes of cold acetone and incubated for 1 h at -20 °C, followed by centrifugation at 12,000 rpm for 10 min. The supernatant fluid was decanted and the pellet was resuspended in 200 μL of rehydration buffer.

For 2-DE analysis of liver lysate prepared by ultrafiltration, 1 mL lysate was dispensed into an Amicon® Ultra-4 10,000 MWCO centrifugal device. The device was spun at 3000 x g for 40 min until approximately 100 μL of concentrated volume was remaining in the device. Nine-hundred microliters of 8 M urea, 2% CHAPS solution was added to the device, and centrifugation was repeated for 40 min. The concentrated proteins were immediately transferred to a microcentrifuge tube and the volume was adjusted to 1 mL with IPG rehydration buffer (8 M urea, 2% CHAPS, 0.002% bromophenol blue, 40 mM DTT, and 0.5% Pharmalyte). One-hundred fifty microliters of reconstituted ultrafiltrate were used to rehydrate an IPG strip in 200 μL total volume.

Results

Bovine liver lysate was prepared by homogenization in isotonic buffer with protease inhibitors. The protein concentration in the extract was determined to be 5 mg/mL. In order to successfully analyze the sample by 2-DE, it was estimated that approximately 0.75 mg of total protein was required, and thus 150 mL of the lysate was prepared in 200 mL volume by addition of 50 mL IPG rehydration buffer (20% of total volume).

2-DE of neat lysate resulted in poor separation and resolution, opresumably because of the high salt concentration and low concentration of chaotropic agents (Figure 1A). To achieve better separation, various methods of sample concentration were attempted. First, the sample was lyophilized and resuspended in IPG rehydration buffer. The resulting 2-D gel was streaky with no well-defined spots and with protein retained at both ends of the IPG strip (Figure 1B).

Acetone precipitation is another method for protein concentration. Figure 1C shows a 2-D gel of acetone-precipitated liver lysate. Because the salt content was greatly reduced compared to Figures 1A and 1B, a satisfactory separation was achieved. While protein recovery appeared to be complete, evaluating this assumption required an alternative method for protein concentration and desalting/ Ultrafiltration (UF) is a well-known method for protein concentration and desalting. Bovine liver proteins were concentrated by centrifugal UF. In this process, proteins are retained and thereby concentrated, while the salt concentration does not change and is the same in the retentate and the ultrafiltrate. However, because of the reduced volume of the retentate, the salt-to-protein ratio in the retentate is lower, and the sample is easily rendered suitable for 2-DE after dilution with IPG buffer.

Figure 1D shows a 2-D gel of liver proteins prepared by UF. As expected, the separation was successful, resulting in multiple well-defined spots on the 2-D gel.

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1. 2-DE gel of 0.75 mg (in 200 μL total volume) bovine liver lysate prepared (A) by mixing proteins with IPG rehydration buffer (B); by lyophilization and resuspension in IPG rehydration buffer (C); by acetone precipitation (C), and (D) by UF. Results show that using ultrafiltration to concentrate a 2-DE lysate sample yields better resolution and higher protein recovery without detectable loss of low-molecular-weight species.

Reference:

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Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression and drug response. Serum is a key source of putative protein biomarkers. One major impediment to discovering new biomarkers is the presence of salts, proteins and lipids in plasma or serum, which interferes with peptide analysis by mass spectrometry. Multiple protocols can be used to extract and enrich peptides from tissues and body fluids:

• After protein precipitation, fractionate by reversed-phase chromatography over C18 resin
• Acetonitrile precipitation of large proteins, while enhancing the solubility of smaller proteins and peptides
• Ultrafiltration for preparing low molecular weight fractions for biomarker analysis^1–4

Ultrafiltration, in combination with solid phase extraction (SPE) on C18 resin is a convenient and efficient method for serum peptide purification. This approach provides more peptides for mass spectrometry analysis when compared to acetonitrile precipitation method. In addition, filter-aided sample preparation (FASP), which was originally developed using our Microcon® centrifugal filters⁵, has recently been optimized for use on Amicon® Ultra centrifugal filters⁶.

One advantage of FASP is that samples can be buffer-exchanged and concentrated through multiple spins without drying/precipitation concerns. Furthermore, FASP was adapted to 96-well plate format using our MultiScreen® Filter Plates⁷. Adaptation to the 96-well format permitted fast processing of biological material and opened possibilities of automating the FASP process in the future.

Methods

I. Preparation of serum peptides II. Peptide analysis by mass spectrometry using the filtrate of Amicon® Ultra 4 mL 10kDa centrifugal ultrafilters III. Preparation of Serum Peptides by Acetonitrile Precipitation (control samples)

Results

Amicon® Ultra-4 30K NMWL centrifugal devices can be used for preparing serum peptides for analysis by high resolution mass spectrometry. When compared to unprocessed rat serum, data quality is improved after removing large proteins by acetonitrile precipitation (Figure 1A, B, below). The number and quality of MALDI-TOF detected peptide peaks was significantly improved using the ultrafiltered serum (Figure 1B, below).

Data quality was further improved by incorporating reverse phase chromatography in the sample preparation protocol, either alone, or in combination with acetonitrile precipitation and ultracentrifugation (Figure 2, below). Here, the ZipTip® μC18 pipette tip served as a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. The highest signal, signal-to-noise ratio and number of detected peptides was achieved with samples prepared using all three methods (Figure 2D, below).

This approach can be used directly in combination with ZipTip® μC18 pipette tips for peptide identification by MS/MS, or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. These protocols may be applicable to other low molecular weight markers, such as drugs and metabolites.

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