Elimination of Affinity Reagent Interference for the Mass Spectrometric Detection of Low-Abundance Proteins Following Immunoprecipitation Angela M. Martin,† Ting Liu,‡ Bert C. Lynn,‡ and Anthony P. Sinai*,† Department of Microbiology, Immunology, and Molecular Genetics and Department of Chemistry, University of Kentucky, Lexington, Kentucky 40536 Received August 10, 2007
Abstract: The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of lowabundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins. Keywords: immunoprecipitation • mass spectrometry • streptavidin • biotin • Toxoplasma
Introduction The application of proteomics technology has greatly expanded our ability to address cell biological questions in a manner never before possible. Despite the power of this resource, we are often limited by the amount and purity of material for mass spectrometric analysis. These problems especially come into play when working with protein levels at the threshold of detection by mass spectrometry or when protein samples contain superfluous proteins at concentrations exceeding that of targets. We encountered both these obstacles while attempting to define the protein complement of the parasitophorous vacuole membrane (PVM) of the intracellular parasite Toxoplasma gondii.1 Because this intracellular parasite must develop within a host cell,2 we were immediately faced with a contaminating bulk of cellular proteins. To circumvent this problem, we developed polyclonal antisera directed at the desired PVM fraction of infected cells to use as an affinity reagent (Martin and Sinai, manuscript in preparation). Despite the development of this reagent, the low concentration of PVM protein limited the yield of immunoprecipitated protein to levels beneath serum proteins such as albumin and immunoglobulin (Ig) and precluded the detection of peptide signals in mass spectrometry. This manuscript describes a straightforward approach to eliminate contaminating proteins from affinity reagents such as antibodies to permit the detection and * To whom correspondence should be addressed. Department of Immunology, Microbiology, and Molecular Genetics, University of Kentucky, 800 Rose Street MS 401, Lexington, KY 40536. Phone: (859) 323-6680. E-mail:
[email protected]. † Department of Microbiology, Immunology, and Molecular Genetics. ‡ Department of Chemistry.
4758 Journal of Proteome Research 2007, 6, 4758–4762 Published on Web 11/10/2007
identification of low-abundance proteins in situations where this contamination masks valuable mass spectrometric data. Due to these difficulties arising from the extreme low concentration of PVM proteins, we performed immunoprecipitations using large parasite preparations (1010 parasites derived from 100 heavily infected 10 cm tissue culture dishes of host Vero cells) coupled with harsh elution conditions of 200 mM glycine (pH 1.8) to maximize protein recovery of target proteins off of Ig. Under these conditions, unfortunately, Ig which had been chemically cross-linked to protein A beads prior to immunoprecipitation also leached into the eluate. Additionally, other serum proteins such as albumin that carried through the immunoprecipitation in small amounts were still present in the eluate in amounts equal to or greater than our rare proteins of interest. These proteins concealed the peptide signature of the low-abundance immunoprecipitated proteins in mass spectrometry. Similar problems are commonly encountered when using other affinity reagents such as GSTfusion proteins as bait to characterize protein interactions of interest. To unmask the peptides of our proteins of interest, contaminating serum proteins had to be removed from the immunoprecipitation eluate prior to mass spectrometry without resulting in the loss of target proteins. In this paper, we report a method that we developed to selectively remove affinity reagents such as Ig from eluates prior to mass spectrometric analysis. By combining a classic serum Ig precipitation technique3,4 with biotinylation of Ig to facilitate its capture with streptavidin, we have generated a protocol that is easily applied in the laboratory for efficient purification of the eluate prior to mass spectrometry. This method is equally applicable to other affinity reagents as long as the reagent contains one or more primary amines for biotinylation and biotinylation does not interfere with the reagent’s ability to interact with proteins of interest. Although not all reagents may contain a lysine residue, the presence of a primary amine at the amino terminus of all peptides leads to broad applicability of this approach. Because biotin is a small moiety, any interference among proteins should be minimal in most cases. Finally, because streptavidin has such a high affinity for biotinylated molecules, the capture can be conducted under such conditions that are not likely to lead to subsequent loss of target proteins from eluate due to nonspecific interactions as may be seen when using columns, membranes, or beadbased approaches to purification. The only limitation to this method occurs when naturally biotinylated target proteins are 10.1021/pr070517a CCC: $37.00
2007 American Chemical Society
technical notes
Elimination of Affinity Reagent Interference of interest to the researcher as these will also be targeted by streptavidin capture.
Materials and Methods Preparation of Serum and Immunoprecipitation. Rabbit polyclonal antisera raised against the T. gondii PVM (Martin and Sinai, unpublished) were subjected to caprylic acid precipitation as published.5 The purified Ig was biotinylated as follows: Antibody supernatant was brought to 1 mg/mL in PBS. EzLink Sulfo-NH-LC-Biotin (10 mM) (Pierce, Rockford, IL) was added to Ig to a final molar equivalent of 10:1, incubated on ice for 2 h, and dialyzed overnight against 1x PBS. Biotinylation of Ig was confirmed by resolution on 10% SDS-PAGE, transfer to a nitrocellulose membrane, and detection using streptavidinHRP (1:10 000) (Jackson ImmunoResearch Laboratories) and chemiluminescent substrate (Pierce). Biotinylated Ig was chemically cross-linked to magnetic protein A beads (Dynal) using DMP (Pierce) as directed. For immunoprecipitation, 3 × 108 parasites were lysed for 30 min on ice in RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.5% NaC29H34O4, 0.1% SDS, 0.025% NaN3, protease inhibitor cocktail [Roche]), and insoluble material was removed by 13 000 rpm centrifugation for 5 min in a tabletop centrifuge. The lysate was precleared in 10% protein A sepharose beads for 2 h at 4 °C. Following removal of the protein A beads, 20 µg of biotinylated Ig cross-linked to magnetic protein A beads was incubated with this lysate overnight. The magnetic beads were then washed three times for 5 min in RIPA buffer. Following immunoprecipitation, proteins were eluted in 100 µL of 200 mM glycine (pH 1.8) for five minutes and neutralized using 1 M Tris base. Magnetic streptavidin beads (Dynal, Oslo, Norway) were incubated with eluate for 2 h at 4 °C and removed using a magnet for removal of biotinylated reagent. Western Blot of Parasite Proteins with Modified Antibody. Toxoplasma lysate (20 µg) was separated on a 10% polyacrylamide gel and blotted using equivalent dilutions of the crude antisera (1:2000), the caprylic acid precipitated Ig (1:200), and the biotinylated Ig (1:20). HRP conjugated goat R-rabbit Ig secondary antibody (1:2000) (Jackson ImmunoResearch Laboratories) combined with chemiluminescence (Pierce) was used to visualize bands. Mass Spectrometric Analysis. Samples were prepared for mass spectrometry using tube-gel digestion.6 Briefly, solvents in the protein sample were removed using a Speedvac. The samples were then dissolved in a minimum volume of water and mixed with acrylamide solution (40%, 29:1), 1% ammonium persulfate, and TEMED at a ratio of 14:5:0.7:0.3. The resulting solution was immediately transferred to a glass centrifuge tube for 30 min until the tube gels formed. The tube gels were incubated with water for 10 min, washed with 25 mM NH4HCO3, dried with acetonitrile, and incubated with trypsin at 4 °C for 40 min Trypsin in-gel digestion was performed at 37 °C overnight, and the peptides were extracted from gel pieces and concentrated using a Speedvac. The sample was acidified by the addition of 1 µL of formic acid and subjected to HPLC-MS/MS in the data dependent mode. A Finnigan LCQ (Classic) ion trap mass spectrometer coupled to a Hewlett-Packard HPLC system (1100 series) was used for LC-ESIMS/MS analysis. Samples were injected into a 350 µm (I.D.) × 15 cm laboratory fabricated C18 column for chromatographic separation. The column was first equilibrated with 95% solvent A (0.1% formic acid in HPLC grade water) and 5% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 4 µL/min. After injection, the mobile phase composition was held at 95% solvent A for 5 min
and then was linearly decreased to 90% in 10 min and maintained at that concentration for 10 min. Over the next 10 min, solvent A was increased to 95% and held there for the final 5 min. The tandem mass spectra were searched against a mammalian MASCOT database and Toxoplasma gondii protein database (www.toxodb.org) using MASCOT daemon software. The parameters were set as follows: trypsin was used as the cleavage enzyme with one missed cleavage allowed. Both the peptide tolerance and the MS/MS tolerance were set to 0.8 Da, and oxidized methionine was chosen as a variable modification. MudPIT scoring was chosen, and the ion score cutoff was set to 20.
Results and Discussion Proteomic technology has opened the door to answer complex questions concerning cell biology in a manner never before possible with quick investigation into entire proteomes, organelle proteomics, post-translational protein modifications, protein–protein interactions, and others.7,8 However, this technology is still limited by the ability to generate samples of sufficient abundance and purity for analysis. When considering proteins of low abundance in a system that cannot be biochemically purified, such as the PVM proteome of T. gondii,1 it is often difficult to impossible to obtain sufficiently purified preparations of protein for mass spectrometric identification. To alleviate this problem, we developed unique polyclonal antisera directed against the PVM-enriched fraction of the infected cell (Martin and Sinai, manuscript in preparation). Although this addressed contamination of host cell proteins, the affinity reagent itself proved problematic due to albumin and Ig in the sample. This was despite chemical cross-linking of the Ig to protein A beads prior to their use for immunoprecipitation; the harsh elution conditions (200 mM glycine, pH 1.8) that were required to maximize elution of the lowconcentration proteins of interest led to a small percentage of Ig being leached from the beads. This amount was significant when compared to our target protein concentration. To analyze these proteins, therefore, we created a novel sample preparation taking advantage of classic caprylic acid precipitation of antisera and biotinylation to sufficiently remove these contaminants from eluate prior to analysis (Scheme 1). Preparation of an Affinity Reagent for Capture Does Not Alter Its Binding Properties. To deal with contaminating serum proteins, most notably albumin, the polyclonal antisera were first subjected to caprylic acid precipitation (CAP).3,4 This protocol has been optimized and published for purifying Ig from the serum of a variety of species.5 This treatment effectively precipitated bulk proteins from the antisera leaving intact Ig in the supernatant as shown by silver stain as was expected (Figure 1A). The only detectable protein bands remaining in solution are the Ig heavy-chain subunit around 50 kDa and the Ig light-chain subunit around 20 kDa. Notably, in the CAP treated lane, albumin (Alb, the prominent 60 kDa band in the untreated antisera) and other serum proteins are absent. Following caprylic acid precipitation of the antisera, we biotinylated the Ig using a commercially available biotin reagent at a final molar equivalent of 10:1 and dialyzed the sample overnight against 1X PBS. To test how the manipulation of Ig affected its binding properties, we examined its ability to recognize target proteins by Western blot analysis. The protein bands recognized on a 10% polyacrylamide band were not different between crude antisera (AS) and caprylic acid purified Ig or biotinylated Ig (CAP/B) (Figure 1B). The polyclonal antisera raised against a Journal of Proteome Research • Vol. 6, No. 12, 2007 4759
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Martin et al.
Scheme 1. Preparation of Antisera for Use in Immunoprecipitation and Their Subsequent Elimination from Eluates prior to Mass Spectrometrya
a
Ig is precipitated from antisera using caprylic acid and is biotinylated prior to use in immunoprecipitation. This facilitates its efficient removal from eluate prior to mass spectrometric analyses.
Figure 1. Ability of manipulated antisera to recognize proteins determined using Western blots. (A) Following caprylic acid precipitation (CAP), the starting material for the biotinylation reaction reveals highly purified Ig heavy- (50 kDa) (HC) and light-chain (20 kDa) (LC) with the loss of other serum proteins including albumin (Alb). (B) Western blot analysis reveals that the crude antisera (AS), the caprylic acid precipitated Ig (CAP), and the biotinylated CAP-purified Ig (CAP/B) recognize the same protein bands on a 10% polyacrylamide gel. The dilutions of antibodies were normalized based on volume of starting material as described in the Materials and Methods. Purification results in increased sensitivity as noted by the enhanced detection of several low-intensity bands denoted by asterisks.
complex protein fraction recognized many protein bands over a broad molecular weight range as was predicted in all three instances. In fact, the efficiency of band recognition increased with manipulation as the Ig was purified and concentrated as evidenced by the increasing band intensity; several lowintensity bands (denoted by asterisks in Figure 1B) were more strongly recognized by caprylic acid purified and biotinylated Ig. This modified Ig could therefore still be used to immunoprecipitate T. gondii PVM proteins. 4760
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Figure 2. Identification of serum proteins in mass spectrometric samples following streptavidin capture. An ion chromatogram of eluate without streptavidin capture shows a single large peak corresponding to the Ig protein (A), whereas the same sample following application of the streptavidin capture protocol shows that even sensitive mass spectrometry techniques can no longer detect Ig (B).
Streptavidin Capture Is Capable of Removing Biotinylated Ig from Eluate beyond the Level of Mass Spectrometric Detection. To test the efficiency of biotinylated protein removal by streptavidin capture, increasing amounts of biotinylated Ig were incubated with magnetic streptavidin beads for 2 h, and the material remaining in solution following removal was monitored by silver staining on SDS-PAGE. We found that under our conditions the use of 2 mg streptavidin beads per 100 µg of Ig in 4 mL volume resulted in optimum removal of Ig, as we could no longer detect any heavy or light chains (data not shown). Because silver staining has a detection threshold several orders of magnitude above that of mass spectrometric detection, it was essential to confirm that the streptavidin capture protocol effectively removed all Ig from the sample. Eluates were harvested from biotinylated Ig crosslinked beads and either left untreated or treated with streptavidin beads as above and analyzed by tandem mass spectrometry. As we had seen before in our proteomics efforts, without streptavidin capture, a large peptide with a retention time of 64.13 min corresponding to the 70 kDa Ig protein was present in our ion chromatograms (Figure 2A). However, following streptavidin capture, this peak was entirely absent in the ion chromatogram confirming that we had been able to effectively remove all Ig from the eluate (Figure 2B). When parasite lysate from 3 × 108 parasites is used in immunoprecipitation for detection, the spectra of the sample in the absence of streptavidin capture show a broad range of peptide peaks eluting from 493.2 to 1241.5 min (Figure 3A-I). All these peaks correspond to the Ig protein and were completely devoid of target proteins of interest (Table 1). As these peptides exist across the entire eluted spectrum, it is impossible to see any of the lowerabundance proteins of interest. The above experiment was repeated with the inclusion of the streptavidin capture protocol. Tandem mass spectroscopy analysis of this immunoprecipitated material revealed an entirely distinct MS profile than was previously obtained. Peptides were recovered from elution times of 547.1 to 1207.1
technical notes
Elimination of Affinity Reagent Interference
Figure 3. Mass spectrometric detection of serum proteins versus low-abundance proteins of interest without streptavidin capture. (A) A total ion chromatogram of the protein sample without streptavidin capture analyzed by LC-MS/MS showing a profile corresponding to IgG as confirmed by the identification of several peptides. (B-H) Extracted ion chromatograms of peptides from IgG with m/z 829.5, m/z 493.2, m/z 1241.5, m/z 1174.8, m/z 819.1, m/z 738.2, and m/z 730.5, respectively. (I) MS/MS spectra of peptide NGFIQSLKDDPSQSTNVLEAK identified from the protein sample without avidin capture. Notably, no non-IgG peptides were identified (see Table 1). Table 1. Immunoprecipitates in the Absence of the Streptavidin Capture Protocol which Reveal only IgG Sequencesa peptide sequence
observed
Mr(exptl)
Mr(calcd)
score
EILAEAK LNDAQAPK LNESQAPK EILAEAKK NGFIQSLK KLNDAQAPK KLNESQAPK ADAQQNNFNK ANGTTADKIAADNK DDPSQSANLLAEAK DDPSQSTNVLGEAK DDPSQSANLLSEAK DDPSVSKEILAEAK DDPSQSANVLGEAQK DDPSQSANLLSEAKK NGFIQSLKDDPSVSK NGFIQSLKDDPSQSANLLAEAK NGFIQSLKDDPSQSTNVLGEAK NGFIQSLKDDPSQSANLLSEAK DQQSAFYEILNMPNLNEAQR NGFIQSLKDDPSQSANVLGEAQK EQQNAFYEILHLPNLTEEQR EQQNAFYEILHLPNLNEEQR EQQNAFYEILNMPNLNEEQR FNKEQQNAFYEILHLPNLNEEQR ADNNFNKEQQNAFYEILNMPNLNEEQR ADAQQNNFNKDQQSAFYEILNMPNLNEAQR
774.44 856.72 886.53 451.37 454.59 493.08 508.14 1149.43 696.09 730.04 731.09 738.08 751.70 779.91 802.04 818.13 1174.01 1175.49 1182.36 1191.22 816.30 1237.46 1243.57 1248.78 959.43 1095.60 1176.77
773.43 855.71 885.52 900.73 907.17 984.14 1014.26 1148.42 1390.17 1458.06 1460.16 1474.14 1501.39 1557.80 1602.07 1634.25 2346.01 2348.96 2362.71 2380.42 2445.86 2472.91 2485.13 2495.54 2875.26 3283.78 3527.27
772.43 855.44 885.46 900.53 905.50 983.54 1013.55 1148.52 1388.69 1457.70 1459.68 1473.69 1500.77 1557.73 1601.79 1633.83 2345.19 2347.17 2361.18 2380.11 2445.21 2471.21 2484.20 2495.14 2873.41 3282.50 3526.62
27 25 35 41 41 78 64 31 51 73 70 71 36 77 34 41 68 66 77 103 54 48 48 72 44 50 62
a Peptides in bold indicate those peptides represented in the ion chromatograms. Note the absence of any parasite proteins in the mass spectrometric identification of the immunoprecipitating proteins.
min similar to the spectra seen without streptavidin capture. However, the spectra of these peptides all belong to T. gondii proteins with no trace of Ig (Figure 4A-H, Table 2). This obviously shows that previously masked peptides which constituted the inherent noise when compared to Ig peptide signal are now clearly evident as a signal. These peptides could not be located in the previous spectrum from the sample without streptavidin capture. Even when using 30fold fewer parasites in these IPs than is optimum for peptide
signal recovery in this system, five parasite proteins and no Ig signal were identified with multiple peptide matches when searched against a T. gondii genome and protein database (www.toxodb.org). These data would be inherently lost were it not for the complete removal of antisera proteins and Ig prior to analysis. This protocol is extremely efficient at removing contamination resulting from affinity reagents in preparations of lowabundance proteins prior to mass spectrometry. As it requires Journal of Proteome Research • Vol. 6, No. 12, 2007 4761
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Figure 4. Mass spectroscopic detection of serum proteins versus low-abundance proteins of interest following implementation of the streptavidin capture protocol. (A) Total ion chromatogram of the protein sample with streptavidin capture analyzed by LC-MS/MS showing a pattern distinct from that seen in Figure 3. The identification of selected peptides is presented. (B-G) Extracted ion chromatograms of peptides from the T. gondii IP sample with m/z 1207.1, m/z 799.3, m/z 816.0, m/z 977.5, and m/z 547.1, respectively. (H) MS/MS spectra of peptide DLVVPQLISSPAWQEVTEMLR identified from the protein sample with avidin capture. These peptides correspond to immunoprecipitated proteins from Toxoplasma (see Table 2) and did not correspond to either Ig or other serum components. Table 2. Peptides of Immunoprecipitated Proteins of Interest Identified following Implementation of the Streptavidin Capture Protocol identified protein
score
peptide sequence
observed
Mr(exptl)
Mr(calcd)
serine-threonine phosophatase 2C major surface antigen P30 precursor
50 41
tetrin A protein actin hypothetical protein
28 22 25
DLVVPQLISSPAWQEVTEMLR LTVPIEK TALTEPPTLAYSPNR SIDILKEEEVK VAPEEHPVLLTEAPLNPK MAEPEQSHNNTVRK
1207.11 799.26 815.78 651.90 977.51 547.15
2412.20 798.25 1629.54 1301.79 1953.01 1638.42
2410.26 798.49 1629.84 1301.71 1953.06 1639.77
no special equipment and widely available reagents, it can be applied easily in the laboratory. Further, as biotin reagents are capable of modifying a wide range of proteins, this technique can be equally applied to other affinity captures using reagents other than Ig. Accordingly, this technique has been equally successful in our laboratory using recombinant proteins and GST fusions as bait to identify interacting proteins (data not shown). Because biotin is a small moiety, occasions where it interferes with substrate interaction will be minimal. Further, because the biotin-streptavidin interaction is of high affinity over a wide range of conditions, the capture can easily be conducted in such a manner as to minimize the recapture of targets from eluate. The loss of protein due to nonspecific binding as can be seen when using columns, filters, or other purification methods is also thus minimized using this approach. This protocol and its empirical optimization to diverse experimental situations have broad applicability permitting the mass spectrometric identification of proteins that were previously masked by an affinity reagent.
Acknowledgment. This work was supported by grant AI062826 to A.P.S. from the National Institutes of Health.
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References (1) Martin, A. M.; Liu, T.; Lynn, B. C.; Sinai, A. P. The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border. J. Eukaryot. Microbiol. 2007, 54 (1), 25–28. (2) Tenter, A. M.; Heckeroth, A. R.; Weiss, L. M. Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 2000, 30 (12–13), 1217–1258. (3) Steinbuch, M.; Audran, R. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch. Biochem. Biophys. 1969, 134 (2), 279–284. (4) Russo, C.; Callegaro, L.; Lanza, E.; Ferrone, S. Purification of IgG monoclonal antibody by caprylic acid precipitation. J. Immunol. Methods 1983, 65 (1–2), 269–271. (5) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988. (6) Lu, X.; Zhu, H. Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins. Mol. Cell Proteomics 2005, 4 (12), 1948–1958. (7) Yates, J. R.; Gilchrist, A.; Howell, K. E.; Bergeron, J. J. Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell Biol. 2005, 6 (9), 702–714. (8) Lin, D.; Tabb, D. L.; Yates, J. R. Large-scale protein identification using mass spectrometry. Biochim. Biophys. Acta 2003, 1646 (1–2), 1–10.
PR070517A
