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• W2141627904 abstract "A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-μm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of α1-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces. A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-μm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of α1-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces. MS is the method of choice for identification of peptides in digests of biological samples based on the power of MS to detect the chemically well defined masses of both peptides and their fragments produced by processes such as CID. This high level of structural specificity is also critical in improving peptide (and protein) quantitation because it overcomes the well known problems inherent in classical immunoassays related to limited antibody specificity, dynamic range, and multiplexability. In principle, a quantitative peptide assay using MRM 1The abbreviations used are: MRM, multiple reaction monitoring; SISCAPA, stable isotope standards and capture by anti-peptide antibodies; FA, formic acid; HSA, human serum albumin; LPS, lipopolysaccharide; AAC, α1-antichymotrypsin; Ab, antibody; LBP, LPS-binding protein; ESI, electrospray ionization. detection in a triple quadrupole mass spectrometer should have nearly absolute structural specificity, a dynamic range of ∼1e+4, and the ability to multiplex measurements of hundreds of peptides per sample (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). These properties suggest that MS-based methods could ultimately replace classical immunoassay technologies in many research and clinical applications. An important limitation of present peptide MRM measurements is sensitivity. The most sensitive widely used quantitative MS platforms use nanoflow chromatography and ESI to deliver trace amounts of peptides to the mass spectrometer. However, these processes are limited in the total amount of peptide that can be applied while retaining maximum sensitivity (typically limited to ∼1 μg of total peptide sample, i.e. the product obtained from digesting ∼14 nl of plasma). The lower cutoff for detecting proteins in a digest of unfractionated plasma by this approach appears to be in the neighborhood of 1–20 μg/ml plasma concentration, which would restrict analysis to the top 100 or so proteins in plasma (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). The sensitivity of MS assays can be substantially increased by fractionating the sample at the level of intact proteins, the tryptic peptides derived from them, or both. For example, immunodepletion of the six most abundant plasma proteins, removes ∼85%of the protein mass (2Pieper R. Su Q. Gatlin C.L. Huang S.T. Anderson N.L. Steiner S. Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.Proteomics. 2003; 3: 422-432Crossref PubMed Scopus (348) Google Scholar) and results in an increase of ∼7-fold in the signal-to-noise of MRM measurements of peptides from the remaining proteins after digestion (1Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). Similarly chromatographic fractionation by strong cation exchange provides another major improvement in sensitivity (3Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). However, increased sample fractionation brings with it the disadvantages of increased cost and time, the risk of losing specific components, and the continued requirement for very high resolution (lengthy, low throughput) reversed phase nanoflow chromatography en route to the ESI source. An alternative fractionation approach, used in the SISCAPA method, enriches specific target peptides through capture by anti-peptide antibodies, thus circumventing these disadvantages for preselected targets (4Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (697) Google Scholar). In its initial implementation, SISCAPA used very small (∼10-nl) columns of POROS chromatography support carrying covalently bound rabbit antibodies and provided ∼100-fold enrichment of target peptides with respect to others (4Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (697) Google Scholar). These columns were, like immunoaffinity depletion columns (2Pieper R. Su Q. Gatlin C.L. Huang S.T. Anderson N.L. Steiner S. Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.Proteomics. 2003; 3: 422-432Crossref PubMed Scopus (348) Google Scholar), recyclable many times. However, the potential for sample-to-sample carryover, limitations in the amount of sample digest that could be pumped over nanoaffinity columns at flow rates slow enough to permit peptide binding, and limited flexibility in changing and multiplexing antibodies were problematic. This led us to explore an alternative approach using magnetic beads as the antibody support (5Whiteaker J.R. Zhao L. Zhang H.Y. Feng L.C. Piening B.D. Anderson L. Paulovich A.G. Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers.Anal. Biochem. 2007; 362: 44-54Crossref PubMed Scopus (234) Google Scholar). In this case, the binding reaction can be carried out off line, allowing equilibrium binding; the magnetic beads can be removed from the digest sample and washed; and the bound peptides can be eluted in 96-well plates either manually or using automated equipment such as a KingFisher Magnetic Particle Processor (ThermoFisher). One potential pitfall remains in the handling of eluted peptides. If the anti-peptide antibodies have very high selectivity, as desired in the SISCAPA approach, then in the case of low abundance peptides, only a very small amount of peptide will be eluted from the antibody. Such small amounts of peptide are easily lost through irreversible binding to the walls of vessels such as 96-well plate wells, and the smaller the amount of peptide (i.e. the more specific the capture), the worse the problem may be. To address this issue, we report here a hybrid approach in which peptide binding occurs off line (to equilibrium), whereas the subsequent washing and elution steps are carried out within a capillary that forms part of the nanoflow LC system, thus ensuring that peptide eluted from the antibodies on the beads will not be “lost” between elution and the ESI source. Although there is extensive literature on macroscopic and microfluidic devices for manipulating magnetic beads (6Safarik I. Safarikova M. Magnetic techniques for the isolation and purification of proteins and peptides.Biomagn. Res. Technol. 2004; 2: 7Crossref PubMed Scopus (436) Google Scholar, 7Pamme N. Magnetism and microfluidics.Lab Chip. 2006; 6: 24-38Crossref PubMed Scopus (978) Google Scholar, 8Rida A. Gijs M.A. Manipulation of self-assembled structures of magnetic beads for microfluidic mixing and assaying.Anal. Chem. 2004; 76: 6239-6246Crossref PubMed Scopus (160) Google Scholar) we were unable to find components adaptable to the small scales and high pressures required for integration into nanoflow HPLC. We therefore developed a novel “bead trap” device that satisfies the following requirements: 1) the need to retain beads in a “trap” region against the flow of liquid (loading, wash, and elution buffers for example) in a vessel of capillary dimensions, 2) the need to ensure that beads do not escape from the trap region to contaminate downstream apparatus or columns, 3) the need to ensure that beads are effectively mixed with the flowing fluids (required for efficient washing and elution), and 4) the need to ensure that all beads can be efficiently ejected from the trap region in preparation for a subsequent cycle. The device provides multiple sequential magnetic trapping regions capable of sweeping commonly used 2.8- and 1-μm magnetic beads against liquid flow to prevent escape of beads through the trapping device (i.e. the second downstream trapping zone captures beads swept by the liquid stream past the first trap and so on). In addition, the bead trap device allows the movement of these trapping regions to agitate the trapped bead mass and mix it with fluids flowing past. Finally the device allows reversal of the sweeping action to effectively eject beads from the trap into the fluid stream. The bead trap capillary can be plumbed at various points in conventional nanoflow LC systems (e.g. in place of a sample loop or connecting tube), and the device can be controlled directly by the LC-MS/MS instrument software through contact closures. We show that the bead trap provides an effective method of implementing SISCAPA experiments. Human plasma digest was prepared from blood collected in 10-ml heparinized vials and centrifuged to liberate the plasma component. This plasma was diluted to 5 mg/ml using 25 mm ammonium bicarbonate, denatured with SDS (0.05%final concentration), and reduced with tris(2-carboxyethyl)phosphine (50 mm final concentration) for 30 min at 60 °C. The sample was cooled to room temperature before alkylating with iodoacetamide (10 mm final concentration) for 30 min at 37 °C. The reduced and alkylated plasma sample was digested with Promega sequencing grade modified porcine trypsin at an enzyme:protein ratio of 1:50 for 12 h at 37 °C. Digestion was quenched using Nα-p-tosyl-l-lysine chloromethyl ketone (100 μm final concentration), and the digest was stored at −80 °C. Rabbit polyclonal antisera to a tryptic peptide of human α1-antichymotrypsin (peptide AAC-1; Table I) were made by immunizing rabbits with keyhole limpet hemocyanin conjugates of AAC-1 peptide with added C-terminal GSGC linker as described previously (4Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (697) Google Scholar). The resulting polyclonal antibodies were affinity-purified on an agarose column to which the respective peptide (plus linker) was conjugated. Approximate binding constants (immobilized antibody binding target tryptic peptide from solution) were measured using a Biacore 3000 system as described previously (9Pope M.E. Soste M.V. Eyford B.A. Anderson N.L. Pearson T.W. Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique.J. Immunol. Methods. 2009; 341: 86-96Crossref PubMed Scopus (42) Google Scholar) and yielded values of k a = 1.91e+5 m−1 s−1, k d = 7.25e−4 s−1, and K D = 3.80e−9 m. In addition, rabbit polyclonal antisera were made to a pool of five tryptic peptides of human LPS-binding protein by immunizing rabbits with pooled keyhole limpet hemocyanin-peptide conjugates with C-terminal Cys extensions; the best responding peptide (LBP-1b; Table I) was selected, and the corresponding specific polyclonal antibody was purified on a peptide antigen affinity column as above. Approximate binding constants for this antibody were measured as above, yielding values of k a = 4.13e+5 m−1 s−1, k d = 1.41e−4 s−1, and K D = 3.42e−10 m. Protein G-coated magnetic beads (Dynabeads G, Invitrogen Dynal, catalog number 100.100.04D; 30 mg/ml reported concentration) were used to capture rabbit antibodies from solution.Table IMRM transitions measured and labeled in the figuresPeak labelAbbreviation in textMS1MS2Protein/detergentSequence1467.3660.4Serum albumin (HSA)LCTVATLR2490.8562.3Haptoglobin β chainVGYVSGWGR3AAC-1531.3819.5α1-AntichymotrypsinEIGELYLPK4559.9697.4IgG1 heavy chainFNWYVDGVEVHNAK5HSA-1575.4937.4Serum albumin (HSA)LVNEVTEFAK6598.7732.4IgG2 heavy chainVVSVLTVVHQDWLNGK7599.8849.5Plasma retinol-binding proteinYWGVASFLQK8603.4997.5IgG1/G3/G4 heavy chainVVSVLTVLHQDWLNGK9610.8775.4HemopexinNFPSPVDAAFR10615.9819.5CHAPS detergent11LBP-1b624.3920.5LPS-binding proteinITLPDFTGDLR12751.9836.5Ig κ light chainDSTYSLSSTLTLSK13856.9882.5HemopexinGECQAEGVLFFQGDR14923.01059.5α2-MacroglobulinLLIYAVLPTGDVIGDSAK Open table in a new tab The LC system was loaded with 0.1 m ammonium acetate, pH 7.5 (high flow solvent A); 2%acetonitrile, 0.1%formic acid (nanoflow solvent A); and 98%acetonitrile, 0.1%formic acid (nanoflow solvent B). Autosampler reagent vials were provided loaded with 0.1 m ammonium acetate, 0.5 m NaCl, 0.1%CHAPS, pH 7.5 (“wash”); 70%acetonitrile, 0.1%formic acid (“bead eluent”); and 1%CHAPS (“bead ejection solution”). A simplified SISCAPA protocol was used in which anti-peptide antibody, free in solution, was added to a sample digest and allowed to incubate followed by addition of Dynal protein G beads and a further incubation to allow capture of the antibody on the beads. In general these additions were carried out in round bottom 96-well polypropylene plates, which were shaken on a microplate shaker (Sarstedt Monomixer MM-1) to keep the beads in suspension. Washing of beads and elution of bound molecules were carried out either manually by transfer of solutions between wells while beads were gathered on a side wall by a Novagen Magnetight HT-96 magnet array or in an automated process (using the bead trap described below) in line with the nanoflow LC system. In both approaches, the natural tendency of the beads to stick to plastic and fused silica surfaces was overcome by addition of low concentrations (0.1%) of CHAPS detergent to most solutions aside from the final wash and elution. CHAPS was selected because it elutes late in the reversed phase separation as a single major peak after most peptides and hence is more “mass spectrometer-friendly” than other detergents, most of which are polymeric, yielding many peaks when monitored by suitable MRM profiles. The presence of CHAPS was detected by a characteristic MRM (615.9/819.5). Parameters for bead capture and elution were optimized by using Alexa Fluor 488-labeled rabbit antibody (Invitrogen A11059), crude Alexa Fluor 488-labeled AAC-1 peptide (made by reacting AAC-1 immunogen peptide, including the GSGC extension, with Alexa Fluor 488 amine-reactive tetrafluorophenyl ester (Invitrogen A30005)), and AAC-1 peptide synthesized with an N-terminal fluorescein (Elim Biopharmaceuticals, Inc., Hayward, CA). Fluorescence signals were quantitated in a fluorescence plate reader (SpectraMax Gemini XS; Molecular Devices, Sunnyvale, CA). In initial optimization studies using reference capture reactions containing 1 μg of antibody and 5 μl of Dynal G beads in a 100-μl total volume, ∼70%of the antibody was captured in 4 h. The bead trap prototype used here (Fig. 1) consisted of an aluminum rotor with eight pairs of ¼-inch diameter × 3/16-inch-thick cylindrical rare earth NdFeB permanent magnets (Amazing Magnets, Irvine, CA) that was rotated about its cylinder axis by a reversible, low speed (2 rpm) synchronous motor. The magnet faces were arranged to be approximately co-planar on their upper surfaces, each side-by-side pair (one with north side up and the other with south side up) generating a local region of high field gradient at the point of contact. The direction of motor rotation was controlled by a contact closure signal from a Spark Holland (Plainsboro, NJ) autosampler auxiliary output under software control. A length of 150-μm-inner diameter (360-μm-outer diameter) Teflon capillary tubing (Upchurch Scientific, Oak Harbor, WA) was configured to follow ∼300° of a circle of diameter the same as (and co-axial with) the circle of magnetic trapping regions (i.e. the circle defined by the contact points of the pairs of magnets as they rotate; Fig. 1). This tube was affixed in the appropriate partial circular path by a piece of thin clear adhesive tape on the underside of a tubing carrier plate of clear acrylic plastic. This plate was finally brought parallel to the upper face of the magnets on the rotor and close to them (almost touching the upper surface of the magnets), aligned so that the tube followed the path of the trap regions as the carrier rotated underneath. Alignment of the tubing mount plate parallel to and close to (but not touching) the upper face of the magnet rotor was facilitated by the use of four threaded rods with finger nuts used to force the tubing mount plate toward the upper surface of the magnet carrier against the resistance of four springs. The finger nuts were tightened until a thin sheet of paper could barely be inserted between the magnets and the capillary tubing at any point. Captured magnetic beads congregate where the tube diverges from the circle, i.e. where the effect of the magnets falls off (see supplemental video Beadtrap CIMG1277.avi). The nano-LC system (Fig. 2) consisted of an Eksigent 2-D NanoLC with Spark Holland autosampler and 10-port auxiliary valve supplemented with an additional MX-7900 six-port valve (Rheodyne), two Harvard syringe pumps (one for pretrap diluent addition and one to deliver 50 nl/min degassed 80%isopropanol added to the postcolumn flow just prior to the ESI tip (ensuring stable spray), the prototype bead trap, and a nanomixer (Upchurch N-200). The mass spectrometer was an Applied Biosystems/Sciex 4000 QTRAP controlled by Analyst software v1.4. The bead trap SISCAPA method consisted of four phases: 1) assembly of the SISCAPA binding reaction mixture and incubation, 2) loading and washing beads in the bead trap, 3) elution of peptides from the beads and transfer to the PepMap C18 trap cartridge, and 4) gradient nanoflow chromatography of the peptides and detection by MRM in the mass spectrometer. Phase 1 was carried out off line in round bottom 96-well polypropylene cell culture cluster plates (Costar 3790; Corning Life Sciences, Lowell, MA) by mixing peptide sample (usually the tryptic digest of 5 μl of human plasma); anti-peptide antibody (typically 1 μg) in PBS, pH 7.5, detergent (typically a volume of 1%CHAPS needed to yield a 0.1%final CHAPS concentration); and washed Dynal G beads (typically 5 μl (or 150 μg) of beads/μg of antibody). Prior to use, the Dynal beads were washed three times by dilution into 5 ml of 0.1 m ammonium acetate, 0.5 m NaCl, 0.1%CHAPS, pH 7.5, and vortexing for 30 s followed by magnetic recovery after which the final bead mass was resuspended in a volume of PBS equal to the volume taken from the original product vial. Plasma digest, antibody, and detergent were typically mixed and incubated for 1–8 h followed by addition of beads and further incubation for 1 h on the microplate shaker. The pH of the final mixture (7.5–8.0) was confirmed by spotting 1 μl onto pH paper. Beads suspended in sample were transferred to snap cap vials for autosampler injection and resuspended manually just prior to injection. In phase 2, a suspension of beads in sample digest (typically 5 μl) was aspirated (sandwiched between two 5-μl volumes of wash solution) into a 10- or 15-μl sample loop mounted across the autosampler injection valve and delivered into the bead trap at 10 μl/min followed by a second 10-μl injection of wash solution. The MX7900 valve directed outflow from the bead trap to waste. Although it is unconventional to aspirate particle suspensions through an autosampler sample probe and injection valve, we observed no evidence of probe clogging and minimal particle retention in the valve. In phase 3, 10 μl of bead eluent (70%acetonitrile, 0.1%FA) was injected through the bead trap at 1 μl/min, and after a 6-min delay, the MX7900 valve was switched to deliver bead trap output to the auxiliary valve and then to the mixer where the eluted peptides in 70%acetonitrile, 0.1%FA were combined with a 10 μl/min flow of 0.1%formic acid from a diluent syringe pump, resulting in eluted peptides in a final concentration of 6%acetonitrile flowing over the PepMap C18 trap cartridge for 7 min. Following transfer of peptides to the PepMap C18 trap, the MX7900 valve was switched back to waste, and a 10-μl volume of bead ejection solution was injected over the bead trap while reversing the direction of bead trap rotor rotation (to sweep in the same direction as flow) to send the beads to waste. Phase 4 was triggered at the beginning of phase 2, and after a 13-min delay, the auxiliary valve was switched to place the PepMap C18 trap (now loaded with enriched peptides) in line with the nanoflow gradient system, which delivered a linear gradient of 5–60%B at 300 nl/min over 20 min followed by a 2-min ramp to 80%B and then re-equilibration. A large series of MRMs covering the peptides of interest as well as a series of strong peptides from high abundance plasma proteins were measured with 10- or 15-ms dwell times. For comparison with unfractionated plasma, a similar protocol was developed in which a plasma digest diluted in 70%acetonitrile, 0.1%formic acid (no antibodies or magnetic beads) was injected directly through the bead trap and diluted through the mixer onto the PepMap trap cartridge. An off-line elution process was used for comparison with the bead trap. Phase 1 of the off-line process was as described above, but elution was accomplished in round bottom polypropylene 96-well plates using the reagent addition steps interspersed with bead collection with a Novagen magnet array followed by resuspension on a Sarstedt plate shaker on “microplate” setting. The protocol proceeded as follows: remove digest; wash four times in 100 μl of 0.1 m ammonium acetate, 0.5 m NaCl, 0.1%CHAPS, pH 7.5; wash one time in water; elute in 20 μl of 5%acetic acid. The 20-μl peptide eluate was placed in empty wells and dried down in a SpeedVac vacuum concentrator after which the peptides were redissolved in 20 μl of 2%acetonitrile, 0.1%formic acid and transferred to clean vials in preparation for 15-μl LC-MS/MS injection. Peak areas of measured MRMs were computed using either Analyst or MultiQuant software (Applied Biosystems). MRMs for peaks labeled in the figures are shown in Table I. A “target relative abundance” was computed as the ratio within a run of the areas of the target peptide peak and either a single high abundance peptide (e.g. HSA-1) or else the total ion current reconstructed as the sum of all MRMs measured. Because we used different sets of MRMs in various experiments, we used the ratio of target to HSA-1 (measured in all experiments) unless otherwise noted. The SISCAPA enrichment factor was computed as the ratio of target relative abundances in a SISCAPA experiment versus an LC-MS/MS run of the same unfractionated plasma digest used in the SISCAPA capture. The ideal eluent to recover the bound peptides would 1) release peptide from antibody, 2) retain the antibody on the bead and prevent its entry into the C18 trap or analytical column, and 3) prevent binding of the peptide to tubing or valve surfaces between the bead trap and the C18 trap. A series of eluents (0.1%HCl, 0.15 m NaCl; 6 m guanidine HCl; and 0.1%TFA plus 0–70%ACN in 10%increments with water alone as a negative control) was tested in comparison with the conventional 5%acetic acid eluent in experiments carried out with Alexa Fluor 488-labeled rabbit IgG (Invitrogen) bound to Dynal protein G beads in 96-well black polypropylene plates processed using the KingFisher automated magnetic bead platform (see supplemental bar graphs). We observed that either 0.1%HCl in 0.15 m NaCl or 0.1%TFA eluted slightly more labeled IgG signal than 5%acetic acid, whereas increasing concentrations of ACN in 0.1%TFA eluted progressively less IgG. The highest ACN concentration tested (0.1%FA, 70%ACN) appeared to prevent release of ∼90%of the antibody from the protein G beads (probably by “crashing” or precipitating the Ab on the bead surface). The acidic pH of all these eluents is expected to release bound peptides from the antibodies based on wide experience with elution of many antigens (e.g. release of major plasma proteins (2Pieper R. Su Q. Gatlin C.L. Huang S.T. Anderson N.L. Steiner S. Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.Proteomics. 2003; 3: 422-432Crossref PubMed Scopus (348) Google Scholar) and tryptic peptides (4Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (697) Google Scholar) bound to immobilized antibody columns) and is confirmed in the experiments reported here: 70%ACN, 0.1%TFA efficiently released Alexa Fluor-labeled AAC peptide from the beads (where it was bound by unlabeled anti-AAC peptide antibody), whereas the Alexa Fluor-labeled Ab is not released in the same eluent. Based on this crash effect and the likelihood that most peptides would not bind to tubing walls in this solvent, we elected to use 70%ACN in 0.1%FA as the standard eluent. To allow the peptides to bind to the C18 trap of the LC system, a 1 + 10 dilution of the eluted peptides with 0.1%formic acid was carried out in a nanomixer immediately before flow over the C18 trap (Fig. 2). Water alone fortunately does not elute either peptide or antibody from protein G beads and thus can serve as a final wash before elution. The rotating magnetic bead trap was tested at a variety of flow rates and bead loads in the presence of 0.1%CHAPS detergent. The action of sweeping successive magnetic trapping regions against the direction of fluid flow (i.e. upstream) was found to trap and retain the nominal bead load (5 μl of the standard Dynal G package concentration of ∼30 mg/ml) at flow rates up to 50 μl/min. A video clip of 5 μl of Dynal G beads maintained in a capillary against a 10 μl/min flow (from left to right) is included as supplemental video Beadtrap CIMG1277.avi. Larger amounts of beads (up to 15 μl) could be retained although with less efficient mixing as the capillary in the primary magnetic trap region was packed with a “solid” but undulating mass of beads. In the absence of detergent, a portion of the bead load formed a more flocculent mass (instead of the smoothly flowing mass seen with detergent) and occasionally adhered to the walls of the bead trap capillary. However" @default.
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• W2141627904 date "2009-05-01" @default.
• W2141627904 modified "2023-10-17" @default.
• W2141627904 title "SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device" @default.
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• W2141627904 doi "https://doi.org/10.1074/mcp.m800446-mcp200" @default.
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