Technical Note pubs.acs.org/ac
Rapid Protein Digestion and Purification with Membranes Attached to Pipet Tips Wenjing Ning and Merlin L. Bruening* Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States S Supporting Information *
ABSTRACT: This paper presents rapid protein purification and proteolysis methods that integrate membrane technology and pipet tips. Pushing a protein-containing solution through a protease-modified membrane at the end of a pipet tip digests proteins in 30 s or less, and the short proteolysis time avoids reformation of disulfide bonds to enable tryptic digestion without alkylation of cysteine residues. Moreover, proteolysis is more complete than digestion for 30 min in solution. Antibody digestion at the end of a pipet tip leads to 100% peptide coverage in MS analyses. Similarly, when membranes contain Ni2+ complexes, pipetting aqueous polyhistidine-tagged protein through the membrane and subsequent rinsing and elution yield purified polyhistidine-tagged protein in 2 min. These applications demonstrate the potential for combining functional membranes and pipet tips for rapid sample purification and pretreatment.
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To interface membranes and pipets, we employ a commercial flangeless ferrule20 as a membrane holder that fits on the end of a 200 μL pipet tip. Using a mechanical pipet, passage of 100 μL of protein-containing solution through a functionalized membrane takes less than 30 s, and proteolysis of apomyoglobin is more complete than in-solution digestion for 30 min and is comparable to digestion in MonoTip trypsin pipet tips with 20 cycles of aspiration and dispensing followed by 20 min of soaking the tip in solution. Digestion of the monoclonal antibody Herceptin in membranes attached to pipet tips also requires less than 30 s and thus avoids the need for protein alkylation prior to tryptic digestion. Finally, we show that a membrane modified with Ni2+ complexes enables purification of His-tagged small ubiquitin modifier (HisSUMO) protein in 2 min when using pipet tips for fluid flow.
rotein isolation and digestion are often vital steps in mass spectrometry (MS) studies of protein structures, interactions, and posttranslational modifications.1−4 Conventional in-solution digestion with a low protease-to-protein ratio usually takes hours, but immobilization of proteases at high concentrations on solid supports can greatly reduce digestion time.5,6 Isolation of specific tagged proteins from complex mixtures, such as cell lysates, also typically requires 30 min or more, in part because slow diffusion into affinity beads limits the rate of protein capture and elution.7,8 Porous membranes present an attractive alternative to beads because convective flow can rapidly transport proteins or reagents to functional sites.9,10 Using simple layer-by-layer adsorption, our group functionalized a series of membranes for enrichment of phosphorylated peptides,11 purification of polyhistidine-tagged (His-tagged) proteins,12,13 and tryptic or peptic protein digestion.14,15 Purification of tagged proteins exploited a peristaltic pump and a 20 mL cell,13 whereas proteolysis occurred using a syringe pump and a membrane with an exposed area of 0.02 cm2 to enable digestion of low-volume solutions.14 However, these apparatuses are impractical for high-throughput digestion or purification. This paper describes membrane-based devices that connect directly to pipet tips to enable convenient proteolysis or protein purification that could potentially couple to robotic systems. Comparable devices employ resin-containing pipet tips.16−19 For example, Monotip (GL Science) and DigesTip (ProteoGen Bio) pipet tips contain monoliths or resins for protease immobilization, and PureSpeed Affinity Resin tips (Mettler Toledo) allow purification of His-tagged proteins and antibodies. Compared to resins, flow through membrane pores should enhance digestion, washing, and elution. Moreover, membrane functionalization through layer-by-layer adsorption is simple and convenient. © XXXX American Chemical Society
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EXPERIMENTAL SECTION Materials. Trypsin from bovine pancreas (type I, 12200 units/mg solid), pepsin from porcine gastric mucosa, poly(acrylic acid) (PAA, average molecular weight of ∼100000 Da, 35% aqueous solution), polyethylenimine (PEI, branched, Mw = 25000 Da), poly(sodium 4-styrenesufonate) (PSS, Mw ≈ 70000 Da), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), aminobutyl nitrilotriacetate (NTA), tris(2-carboxyethyl)phosphine (TCEP), and apomyoglobin (protein sequencing grade from horse skeletal muscle, salt-free lyophilized powder) were purchased from Sigma-Aldrich. MonoTip trypsin pipet tips were obtained from GL Science. His-SUMO protein was overexpressed in BL21DE3 cells, and Herceptin (Genentech) Received: September 29, 2015 Accepted: November 6, 2015
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DOI: 10.1021/acs.analchem.5b03679 Anal. Chem. XXXX, XXX, XXX−XXX
Technical Note
Analytical Chemistry
For in-solution apomyoglobin digestion, 100 μL of 0.1 mg/ mL protein in ammonium bicarbonate was mixed with 1 μL of a 0.5 mg/mL trypsin solution to initiate proteolysis with a protease to protein weight ratio of 1:20. The proteolysis was stopped at the desired time by adding 1 μL of acetic acid. Mass Spectrometry and Data Analysis. Digests were analyzed with electrospray ionization (ESI)-MS. A protein digest reconstituted in 50 μL of MS buffer (1% acetic acid, 50% methanol, and 49% water) was loaded into a Whatman multichem 96-well plate and sealed with Teflon Ultrathin Sealing Tape. The samples were introduced into the highresolution accurate mass Thermo LTQ Orbitrap Velos mass spectrometer (San Jose, CA) using an Advion Triversa Nanomate nanoelectrospray ionization source (Advion, Ithaca, NY). The spray voltage was 1.4 kV, and the gas pressure was 1.3 psi. High-resolution mass spectra were acquired in positive ionization mode using the FT analyzer operating at 100000 resolving power with relative intensity as the Y-axis. Peptides were identified manually by comparing the experimental data to m/z values for the theoretical peptides generated using the ProteinProspector MS-Product program (v 5.14.1 University of California, San Francisco). Peaks with relative intensities above 1% were analyzed. Settings for the theoretical peptide generation included tryptic or peptic digestion, a maximum number of 99 missed cleavages, peptide masses from 300 to 50000 Da, a minimum peptide length of 2, and oxidation as a variable modification. Although not appropriate for protein identification, these settings ensure that we obtain the total detected peptide coverage of the protein. His-Tagged Protein Purification from Cell Lysate. Cell lysate containing His-SUMO protein was diluted 1:9 in phosphate buffer (20 mM, pH 7.4), and 25 μL of this diluted cell lysate was passed through the membrane holder. A new tip was then used to pass 100 μL of 20 mM phosphate buffer through the holder to wash the membrane. Finally, using a third pipet tip, 25 μL of elution buffer (20 mM phosphate buffer, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) was passed through the membrane to elute the bound His-SUMO protein. The whole process took ∼2 min. Loading, effluent, and eluate (25 μL each) solutions were mixed with 5 μL of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and loaded onto a 4−20% gradient gel.
was a gift from Dr. Mohammad Muhsin Chisti of Michigan State University. Buffers were prepared using analytical grade chemicals and deionized water (Milli-Q, 18.2 MΩ cm). Membrane Modifications. Nylon membranes with nominal pore sizes of 1.2 μm (Hydroxylated, LoProdyne LP, Pall, 1.2 μm pore size) and 5.0 μm (Sterlitech, NY5025100) served as substrates for immobilization of proteases or metalion complexes. Electrostatic immobilization of trypsin and pepsin followed published procedures that comprise sequential adsorption of PSS and protease.14,15 Membrane modification with PAA/PEI/PAA-NTA-Ni2+ films followed a literature protocol and included adsorption of PAA, PEI, and PAA followed by derivatization with aminobutyl NTA and formation of the Ni2+ complex.13 Membranes were cut to fit underneath the ferrule in a PEEK Super flangeless ferrule module (IDEX, Catalog number: P260) (Figure 1). A 200 μL pipet tip (Denville Scientific) fits
Figure 1. Diagram of the membrane holder and attachment of a pipet tip to the holder.
into the ferrule to enable flow, and the holder exposes a membrane diameter of 1.5 mm, which is equivalent to an external surface area of 0.02 cm2. Protein Digestions in Protease-Modified Membranes, in MonoTip Trypsin Pipet Tips and in Solution. Apomyoglobin (0.1 mg/mL) was dissolved in 5% formic acid (pH 2) or 10 mM ammonium bicarbonate (pH 7) for digestion with pepsin or trypsin, respectively. Herceptin (50 μg) was dissolved in 20 μL of HCl (pH 2.5), and after adding 1 μL of TCEP (0.5 M), the mixture was incubated at 56 °C for 30 min to reduce disulfide bonds. Finally, 479 μL of 5% formic acid or 10 mM ammonium bicarbonate was added to make the Herceptin concentration 0.1 mg/mL for pepsin and trypsin digestion, respectively. Using adjustable volume pipets (VWR Ergonomic High-Performance), pepsin- and trypsin-modified membranes were rinsed with 100 μL of 5% formic acid (pepsin) or 10 mM ammonium bicarbonate (trypsin, pH 7) prior to passage of 100 μL of protein solution through a membrane. Samples were collected in eppendorf tubes and immediately dried with a SpeedVac. Apomyoglobin digestion in MonoTip trypsin pipet tips was performed following the manufacturer’s protocol: aspirating and dispensing 100 μL of protein solution for 20 cycles (2−3 min) using the pipet and then soaking the pipet tip in the protein solution for 20 min.21 One cycle of aspiration and dispensing of apomyoglobin solution in the MonoTip trypsin pipet tip was also performed for comparison to digestion during a single pass of a 100 μL protein solution through a membrane.
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RESULTS AND DISCUSSION Membrane Selection. When choosing membranes for pipet tips, selection of an appropriate pore size is vital to minimize diffusion limitations without creating a large transmembrane pressure drop. With very small pores, the mechanical pipet is not sufficient to force solution through the membrane. Equation 1 approximately describes the pressure drop, ΔP, across a porous membrane. 8μLQ ΔP = (1) εAr 2 In this equation, μ is the dynamic viscosity (∼10−3 Pa s for water), L is the membrane thickness (∼100 μm), Q is the volumetric flow rate through the membrane, ε is the membrane porosity (∼0.5), A is the membrane surface area (0.02 cm2), and r is the radii of membrane pores. This expression assumes cylindrical pores with a tortuosity of 1 and thus likely underestimates ΔP. On the basis of eq 1, the passage of a 100 μL solution through a membrane in 30 s requires 430 and 7400 Pa of pressure for membranes with pore diameters of 5 B
DOI: 10.1021/acs.analchem.5b03679 Anal. Chem. XXXX, XXX, XXX−XXX
Technical Note
Analytical Chemistry and 1.2 μm, respectively. A mechanical pipet can provide these pressures, which are
