SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device

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 for in the amount of sample digest that could be columns at flow to peptide and limited in and antibodies were This to an alternative approach using magnetic beads as the antibody support L. Anderson L. enrichment of peptides on magnetic beads for of serum 2007; PubMed Scopus Google Scholar). In this the binding reaction can be equilibrium the magnetic beads can be from the digest sample and and the bound peptides can be in or using automated such as a in the of the anti-peptide antibodies have very high as in the SISCAPA approach, in the of low abundance a very small amount of peptide be from the small amounts of peptide are through binding to the of such as and the the amount of peptide the specific the the the this a approach in which peptide binding the subsequent washing and elution are a capillary that forms part of the nanoflow thus that peptide from the antibodies on the beads be elution and the ESI source. is on and for magnetic beads M. for the and of proteins and Res. 2004; PubMed Scopus Google and 2006; 6: PubMed Scopus Google of of magnetic beads for mixing and 2004; PubMed Scopus Google Scholar) were to components to the small and high for nanoflow developed a novel that the the to beads in a against the flow of liquid and elution for in a of capillary the to that beads from the trap to or the to that beads are with the flowing for washing and and the to that beads can be from the trap in for a subsequent The provides multiple magnetic regions of used and magnetic beads against liquid flow to of beads through the the beads by the liquid the trap and so In the bead trap allows the of these regions to the bead mass and it with flowing the allows of the to beads from the trap the The bead trap capillary can be at in nanoflow (e.g. in of a sample or and the can be by the LC-MS/MS through that the bead trap provides an method of SISCAPA plasma digest was from in and to the plasma This plasma was to using with and with for at The sample was to with for at The and plasma sample was with at an of for at was using and the digest was at to a tryptic peptide of human α1-antichymotrypsin were by with of peptide with as (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 antibodies were on an to which the peptide was binding antibody binding target tryptic peptide from were measured using a as Anderson N.L. Pearson T.W. antibody of high by a PubMed Scopus Google Scholar) and of a and In rabbit were to a of tryptic peptides of human LPS-binding protein by with with the peptide was and the specific antibody was on a peptide as binding for this antibody were measured as of a and magnetic beads were used to capture rabbit antibodies from measured and in the in albumin albumin in a The was with flow and were provided with and A simplified SISCAPA protocol was used in which anti-peptide in was to a sample digest and to by of protein beads and a to capture of the antibody on the In these were in which were on a to the beads in of beads and elution of bound were by of while beads were on a by a or in an automated the bead trap in with the nanoflow system. In both the of the beads to to and was by of low of to most from the and was because it in the reversed phase as a major after most peptides and is than other most of which are many by MRM The of was by a MRM for bead capture and elution were by using rabbit antibody peptide by the with and peptide with an were in a In initial using capture reactions μg of antibody and of beads in a total the antibody was captured in The bead trap used of an with of diameter that was its by a low The were to be on their with and the other with a of high at the of The direction of was by a from a A of 150-μm-inner diameter capillary was to of a of diameter the as (and the of magnetic regions the defined by the of the of as This was in the by a of on the of a of This was to the of the on the and to the of the so that the the of the trap regions as the of the to and to the of the was by the use of with used to the the of the against the of The were a of could be the and the capillary at magnetic beads the from the i.e. the of the The of an with and with an for and to deliver to the flow to the ESI stable the bead and a The mass spectrometer was an by The bead trap SISCAPA method of of the SISCAPA binding reaction and and washing beads in the bead elution of peptides from the beads and to the trap and nanoflow chromatography of the peptides and detection by MRM in the mass spectrometer. was in by mixing peptide sample the tryptic digest of of human anti-peptide antibody (typically in (typically a of to a and beads (typically of of to the beads were by of and for by magnetic after which the bead mass was in a of to the from the product and were and for by of beads and for on the The of the was by in sample were to for and to In phase a of beads in sample digest (typically was of a or sample the and the bead trap at by a of The from the bead trap to it is to through an sample and of and in the In phase of bead was through the bead trap at and after a the was to deliver bead trap to the and to the the peptides in were with a flow of from a in peptides in a of flowing the trap for Following of peptides to the the was to and a of bead was the bead trap while the direction of bead trap in the direction as to the beads to was at the of phase and after a the was to the trap with enriched in with the nanoflow which a of at by a to and A large series of the peptides of as well as a series of strong peptides from high abundance plasma proteins were measured with or times. For with unfractionated a protocol was developed in which a plasma digest in antibodies or magnetic was through the bead trap and through the the trap An off-line elution was used for with the bead of the off-line was as elution was in using the with bead with a by on a on The protocol as in 100 of in in of The peptide was in and in a after which the peptides were in of and to in for LC-MS/MS of measured were using or for in the are shown in A relative was as the a of the of the target peptide and a high abundance peptide (e.g. or the total as the of used of in used the of target to in The SISCAPA enrichment was as the of target relative in a SISCAPA an LC-MS/MS of the unfractionated plasma digest used in the SISCAPA The to the bound peptides would peptide from the antibody on the bead and its the trap or and binding of the peptide to or the bead trap and the A series of and in with as a was in with the in with rabbit bound to protein beads in using the automated magnetic bead that in or than of in The to of the antibody from the protein beads by or the on the bead The of these is to bound peptides from the antibodies based on with elution of many (e.g. 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 antibody and is in the peptide from the beads it was bound by peptide the is in the on this and the that most peptides would bind to in this to use in as the the peptides to bind to the trap of the a of the peptides with was in a flow the trap peptide or antibody from protein beads and thus can as a The magnetic bead trap was at a of flow and bead in the of The of magnetic regions against the direction of flow was to trap and the bead of the of at flow to A of of beads in a capillary against a flow to is as amounts of beads to could be with mixing as the capillary in the magnetic trap was with a mass of In the of a of the bead a mass of the flowing mass with and to the of the bead trap However, this to washing or elution and was reversed a of was to the of of the bead trap so that the magnetic trap regions in the as the liquid in of the the of washing and elution using protein beads with rabbit antibody that in bound peptide from the bead trap was in a step and subsequent elution with and in a The of the of peptide at is or of the in which the beads can be or with a such as the The bead trap was in an nanoflow LC-MS/MS by it in of a and A SISCAPA enrichment was by the bead trap with a human plasma digest to nl of μg of rabbit peptide and of protein beads in and A total of were by the in triple quadrupole the peptide and a series of peptides other high abundance plasma proteins such as an unfractionated a sample the unfractionated digest of nl of human plasma to in μg of total antibodies or was through the and to capture on the trap to the used for the SISCAPA the results of for both In unfractionated digest peptide a small and the peptide is in SISCAPA the peptide is in and the peptide is to The of to target relative is in the unfractionated digest and in the SISCAPA a in enrichment of relative to of In the SISCAPA the for was increased an of antibody capture of an amount of plasma A tryptic peptide of LPS-binding protein plasma abundance is lower than was captured by an rabbit antibody an than the from the digest of of human SISCAPA enrichment in to be MRM a lower abundance peptide from amounts of plasma digest for SISCAPA a series of to a range of high abundance peptides from the plasma the MRM were also an MRM for and that after bead washing its was was in the unfractionated plasma digest MRM is shown in it was a after antibody capture and bead trap elution The of to was and in the unfractionated plasma and SISCAPA respectively, an enrichment of The was after capture from a capture of plasma of proteins T. lipopolysaccharide-binding protein in 2006; PubMed Scopus Google S. M. H. T. A clinical of serum in and PubMed Scopus Google E. T. of α1-antichymotrypsin and in and with and 2007; PubMed Scopus Google in a The peptides were after the peptides were from the beads with and the peptide was and in for LC-MS/MS The for the peptide after off-line elution was that after bead trap elution using capture to of sample The enrichment for was with The of bead trap off-line elution was in of which such bead trap off-line of the SISCAPA capture The of is of off-line elution and is of the bead trap (e.g. with trap elution using a of peptides very to that with that the of peptides that and off-line elution methods in peptide and the were substantially in off-line processing as to bead trap the bead trap the nonspecifically bound tryptic peptides from high abundance plasma proteins in with a of other anti-peptide antibodies, that these peptides be bound at from the peptide binding of the peptides were bound by the antibodies other than the binding or by other components such as the bead SISCAPA capture reactions using the antibody with an reaction from which antibody was The peptide a was the target peptide as the binding are with the of the magnetic beads or capillary than the capture antibody the of mass spectrometry to of proteins is a major of Quantitative absolute specificity and large with MS sensitivity an with the immunoassays detection of For a protein the of MRM quantitation is to of the protein or of a tryptic peptide derived from the the digest of of plasma could be such an LC-MS/MS this would an sensitivity of to the immunoassays and most known nanoflow ∼1 μg of total peptide to the tryptic digest of nl of such an to digests of unfractionated are thus of of the sensitivity depletion (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 strong cation exchange fractionation (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) can sensitivity by per fractionation as can other fractionation R. Gatlin C.L. Su Q. M. Huang S.T. R. Anderson N.L. Steiner S. The human serum of nearly protein on and identification of 2003; 3: PubMed Scopus Google M. A step fractionation approach for plasma using immunodepletion of abundant proteins and chromatography: to the analysis of and PubMed Scopus Google L. R. L. E. based on stable isotope and Mass PubMed Scopus Google R. H. of from 2007; PubMed Scopus Google L. Q. H. M. M. S. of protein fractionation to of analysis of the serum and plasma Proteome Res. 2007; 6: PubMed Scopus Google Scholar), MRM measurements protein These methods have the of to large of plasma or and depletion the of tryptic digestion The disadvantages are the of such methods increased cost and of and the increase in the of LC-MS/MS that be to an of of than such to sensitivity is for analysis of large clinical sample and thus the of or The SISCAPA (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) method a approach, using antibodies) to target peptides from a digest (e.g. plasma). on magnetic L. Anderson L. enrichment of peptides on magnetic beads for of serum 2007; PubMed Scopus Google Scholar) of this method because such use capture the of inherent in the capture from of digest than can be a of a sample digest for capture of and off-line to many samples in for the reaction to equilibrium In this target peptides from a digest of or 100 of plasma should or improvement in MRM sensitivity with unfractionated digest by the plasma of the LC-MS/MS sample from nl to digests are cost and from sample of and 100 of plasma would of of the sensitivity of high clinical in triple quadrupole MS sensitivity should this a of to the for MS to replace clinical immunoassays while specificity with magnetic bead of SISCAPA to maximum and by of antibody to the This approach allows mixing of antibody and is well to antibodies magnetic beads with protein are with the antibody or to bind the antibodies from and and their peptide of the digest for subsequent washing and In a capture that μg of antibody to capture most peptide targets from a can be with high by this capture A with this approach is the of the antibodies from the beads with the peptides on elution and of of the trap or are to such a the antibodies could be at the cost of in to be bound by a trap could be used that is of binding peptides proteins, to the used by and of from by immunoaffinity liquid PubMed Scopus Google Scholar) to bind small elution could be to peptides from the antibodies the antibodies from the beads, which is the that the and that of high (e.g. the antibodies on the In this approach the of peptides from the magnetic bead to the the with a that prevents peptide would the peptides from binding to chromatography a step to the specific SISCAPA the peptide sample for MS in a of hundreds of of peptides to a This results in of and for reversed phase chromatography (and increased sample that for peptides of in an unfractionated plasma digest is in an in total sensitivity for The for the reversed phase chromatography step (e.g. from the to is an the of which to be The of target peptides from a digest at the risk of losing 100 or of a target peptide is very to a to binding The well known of peptides from digests of small is to such on the of Similarly the of of a peptide from a is to be have in the SISCAPA method at peptide after used a magnetic bead trap to loss of peptides captured by anti-peptide antibodies on magnetic carrying the washing and elution inside a capillary flow that can be by magnetic regions against the direction of liquid flow and by that a continuous succession of trap regions are to capture beads from the trap the can a mass of magnetic beads in a 150-μm-inner diameter capillary while liquid by at to of the trap regions also the beads with this could bound peptides in a which is than the in magnetic bead The and direction of and is with LC-MS/MS through The remaining digest peptides can be in the sample for subsequent capture of other In with antibody and captured from an unfractionated human plasma obtained enrichment of 1,800 and respectively, a high abundance albumin These an of the enrichment by the antibody because the like the other was through the capture by with the beads, the In this binding could be it appears A and that the target peptide the measured would a the for of the target The approach can be improved by it is that magnetic beads with peptide binding are because peptide is bound nonspecifically than the beads have developed to protein as in to peptide is that the beads used on a and thus are to bind a of beads (e.g. or would also be because would in and capture or the bead A for development is the flow system. the bead trap nanoflow a for and elution would and method In the it should be to the for reversed phase to peptides and on very at a peptides very or ultimately to the reversed phase by the SISCAPA capture the mass spectrometer. The important for development binding suggest that capture of low abundance peptides from large digest antibodies with of The and rabbit used have measured of and respectively, with the enrichment have or with a of rabbit antibodies specific for peptide targets Anderson N.L. Pearson T.W. antibody of high by a PubMed Scopus Google Scholar), that the is to be The bead trap approach provides a magnetic fractionation methods small of and the nanoflow LC-MS/MS quantitation and absolute structural and in T. for measurements of antibody for of MS The bead trap was developed and by Anderson with