Enrichment of EGFR/PI3K/AKT/PTEN Proteins for Research using Immunoprecipitation and with Mass Spectrometry-based Analysis

Transcription

1 Enrichment of /PI3K/AKT/ Proteins for Research using Immunoprecipitation and with Mass Spectrometry-based Analysis Bhavin Patel, Scott Meier, Kay Opperman, Paul Haney, Barbara Kaboord, John Rogers Thermo Fisher Scientific, Rockford, IL, USA

2 Overview Purpose: Identification and quantification of /PI3K/AKT/ proteins for research using an optimized immunoprecipitation to mass spectrometry (IP-MS) workflow. Methods: We evaluated immunoprecipitation with directly coupled antibodies or biotinylated antibodies with immobilized streptavidin resin., PI3K, AKT isoforms and were enriched from two cell lysates using an optimized IP to MS workflow. A multiplex, targeted selected reaction monitoring (SRM)-based MS research method was developed to measure the limit of quantitation (LLOQ) of,, AKT1,, PIK3CA and tryptic peptides. Multiple targets (, AKT isoforms, ) were immunoprecipitated simultaneously and quantified by targeted SRM assay. Results: Immunoprecipitation using magnetic beads resulted in overall higher yield of target protein and less non-specific binding than agarose beads for MS research applications. Enrichment of,, AKT1 and from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant in human plasma matrix allowed absolute quantitation by LC-SRM. Multiplexed target immunoprecipitation resulted in simultaneous identification and quantitation by MS. Introduction A major bottleneck in the verification of protein biomarkers in clinical research is the lack of methods/reagents to quantify medium to low levels of proteins of interest in human samples. Immunoprecipitation (IP) and mass spectrometry (MS) are complementary techniques that permit sensitive and selective characterization and quantitation of low abundance protein analytes in cell lines, tissue, and biofluids. IP provides both enrichment and high selectivity while the MS provides high selectivity, sensitivity, and multiplexing across a range of analyte concentrations in different matrices. The quantitative evaluation of protein expression and PTM status of - PI3K-AKT signaling pathway proteins enables the precise characterization of the disease. FIGURE 1. Enrichment is necessary for medium to low abundant proteins. Methods Sample Preparation from lysate was immunoprecipitated by direct IP methods (Hydrazide activated polyacrylamide bead, Aldehyde activated agarose bead, NHS-Ester activated magnetic bead, and Epoxy activated magnetic bead) and indirect IP methods (Streptavidin coated polyacrylamide bead and Thermo Scientific Pierce Streptavidin magnetic bead). IP eluted samples were evaluated by western blot and in-solution trypsin digestion followed by MS analysis. IP conditions were optimized for enrichment of medium to low abundant targets (, AKT isoforms, and PIK3CA) for MS applications. Multiplex IP was performed to enrich, AKT isoforms and targets simultaneously from lysate with biotinylated antibodies and with Pierce Streptavidin coated magnetic beads. Liquid Chromatography and Mass Spectrometry IP eluates were reconstituted in 6M Urea, 50mM Tris-HCl ph 8 followed by reduction, alkylation and trypsin digestion overnight. Prior to MS analysis, tryptic digest samples were desalted using the Thermo Scientific Pierce C18 Spin Tips. For discovery MS, the samples were analyzed by LC-MS/MS using a nanolc system at 300 nl/min over a 45 min gradient and Thermo Scientific Orbitrap XL mass spectrometer (DDA, Top 6, CID). For targeted MS, the samples were analyzed by LC-SRM/MS with the Thermo Scientific TSQ Vantage mass spectrometer and Thermo Scientific Easy nanolc II system. Data Analysis Discovery MS data were analyzed with Thermo Scientific Proteome Discoverer 1.4 and Scaffold 4.0 software to assess percent sequence coverage, spectral counts and PTMs. For targeted LC-SRM/MS data analysis, Thermo Scientific Pinpoint and Skyline software were used to measure limit of quantitation (LOQ ) and target analyte concentration. 2 Enrichment of /PI3K/AKT/ Proteins for Research using Immunoprecipitation and with Mass Spectrometry-based Analysis

3 Results FIGURE 2. Experimental workflow for IP-MS research method development. Protein targets are immune-enriched from matrix and analyzed by silver stain or Western blot after gel electrophoresis. IP samples are also digested with trypsin and analyzed by to identify candidate quantitative peptides. Heavy isotopelabeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation. FIGURE 3. Evaluation of immunocapture efficiency and selectivity. ELVE Anti- Western In-Solution Results Heavy chain Success Criteria: <60 >60% P: Polyacrylamide, A: Agarose M: Magnetic + Anti-; - Rabbit IgG immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) sequence coverage and background proteins were determined by LC-MS/MS after elution and trypsin digestion. IP using magnetic beads resulted in fewer background proteins identified and higher sequence coverage. Benefits of Magnetic Beads for IP-MS Lower background: Minimal non-specific binding High signal to noise: Easy and efficient washing, less void volume reduces the chance of losing sample Easy handling: Easy separation of resin Time and effort: Less washing and faster incubation (60 minutes start to finish) Better reproducibility: Product and handling consistency savings: All binding on outer surface Automation: Improves throughput and reproducibility Thermo Scientific Poster Note PN ASMS-EN-0614S 3

4 FIGURE 4. Identification of multiple phosphorylation sites for peptides. A 1166-Phosphoserine 991-Phosphoserine 693- Phosphothreonine by PKD/PRKD1 Immunoprecipitation to tar After enrichment by IP, SRM proteins in the low fmo FIGURE 5. Detection and q All six targets were monitore and peptides were q FIGURE 6. Quantitation of and peptides. Target Peptide No. AKT1 PIK3CA 451-Phosphothreonine B Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\\Batch2\IP_PMS R1_1.raw #2257 RT: ITMS, CID@35.00, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.5 Da ELVEPL(pT)PSGEAPNQALLR 25 y₁₅² Intensity [counts] (10^3) b₃ y₃ A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for and peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of. Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads % Sequence Coverage b₄+-h₂o y₄ y₅ y₁₃²+-p Target y₇ y₁₅²+-p, y₁₄² [M+2H]²+-P y₈ Anti-target y₁₂ Negative Control y₁₃+-h₂o b₁₁+-p b₁₁+ b₁₂ y₁₄+-p y₁₃ y₁₅+-p y₁₅ y₁₆+-h₂o Anti-target b₁₇ b₁₈+-p b₁₈ m/z Negative Control 65% 0% 16% 0% AKT1 36% 2% 68% 6% 50% 0% 82% 0% AKT3 8% 0% 62% 0% 16% 0% 36% 0% PIK3CA 0% 0% 0% 0% -AKT pathway targets were immunoprecipitated from two cell lines with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads, washed, eluted, digested in-solution, and analyzed by LC-MS/MS to assess sequence coverage and identify isoform-specific peptides. Enrichment of from tw peptides by targeted MS. Be Magnetic beads compared to FIGURE 7. Recovery of rec ( % Sequence Cover r Mono P A 0 ng 0% 0 7 ng 3% 2 36 ng 20% ng 42% 2 r spiked into 1mg plas Monoclonal antibody recove FIGURE 8. Multiplex immu Targets/ lysate % Sequen Coverag 17% 23% AKT1 16% 11%, AKT isoforms and PT biotinylated antibodies, capt targets were identified and q 4 Enrichment of /PI3K/AKT/ Proteins for Research using Immunoprecipitation and with Mass Spectrometry-based Analysis

5 QALLR Immunoprecipitation to targeted MS research application () After enrichment by IP, SRM assays enabled the quantitation of,, AKT1, proteins in the low fmol range. FIGURE 5. Detection and quantitation limits of,, AKT1,, PIK3CA and peptides. Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R 2 ) AKT PIK3CA All six targets were monitored with linear quantification.,,, PIK3CA and peptides were quantified from 3.9 fmol to 1000 fmol. FIGURE 6. Quantitation of peptides by targeted MS. Enrichment of from two cell lysate allowed for quantitation of two unique peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads. FIGURE 7. Recovery of recombinant (r) from plasma matrix. ( % Sequence Coverage) r Mono Poly 0 ng 0% 0% 7 ng 3% 2% 36 ng 20% 12% 180 ng 42% 26% Pierce Streptavidin Magnetic Beads Dynabeads MyOne Streptavidin T1 ( Peptide Quantitation) r spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more r. FIGURE 8. Multiplex immunoprecipitation to MS research applications. Targets/ lysate % Sequence Coverage 17% 23% AKT1 16% 11% Targets/ lysate (fmol) (ng) AKT1 >ULOQ >ULOQ , AKT isoforms and were enriched simultaneously from lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS. FIGURE 9. Summary of -AK two cell lines without and with e Target AKT1 AKT3 Grp94 PIK3CA Cell line Conclusion Immunoprecipitation using resulted in a higher yield o using directly immobilized Enrichment of, AKT lysates enabled detection Immunoprecipitation of EG of multiple isoforms and ph, AKT1, and P by in two ce Enrichment of as low as 7n of recombinant PIK3CA/PI absolute quantitation by ta Mulitplex IP to MS allowed,, AKT1 and P Future work will focus on o abundant targets (<1ng tot References Det N 1. Logue JS, Morrison DK. Com use of targeted inhibitors in c Gingras AC, Gstaiger M, Ra using mass spectrometry. Na 3. Carr SA, batiello SE, Acke Biology and Medicine: Best P Development Using a Fit-for 13(3): Ackermann BL. Understand spectrometry methods for cl 58(12): Scaffold is a trademark of Proteome Software and its subsidiaries. For Research use only. Not for This information is not intended to encourage intellectual property rights of others. Thermo Scientific Poster Note PN ASMS-EN-0614S 5

6 plication () ntitation of,, AKT1, GFR,, AKT1,, PIK3CA (fmol) ULOQ (fmol) Linearity (R 2 ) ion.,,, PIK3CA to 1000 fmol. geted MS. Streptavidin Magnetic Beads ads MyOne Streptavidin T1 293 r quantitation of two unique ved with Pierce Streptavidin (SA) treptavidin T1 beads. R) from plasma matrix. FR Peptide Quantitation) ntitated at >7ng (52 fmol). research applications. e (fmol) (ng) >ULOQ >ULOQ ltaneously from lysate with vidin coated magnetic beads. All four FIGURE 9. Summary of -AKT pathway targets identified and quantified in two cell lines without and with enrichment. Target AKT1 AKT3 Grp94 PIK3CA Conclusion Immunoprecipitation using magnetic beads for MS research applications resulted in a higher yield of target protein and less non-specific binding than using directly immobilized antibody. Enrichment of, AKT isoforms, and in and lysates enabled detection by discovery MS and quantitation by targeted MS. Immunoprecipitation of and resulted in simultaneous analysis of multiple isoforms and phosphorylation sites., AKT1, and were quantified in the low nanogram range by in two cell lysates. Enrichment of as low as 7ng recombinant in plasma matrix and 10ng of recombinant PIK3CA/ in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS. Mulitplex IP to MS allowed simultaneous detection and quantification of,, AKT1 and targets. Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein). References Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP N/A N/A - + N/A N/A + + N/A N/A + + N/A N/A - - N/A N/A - - N/A N/A - - N/A N/A - - N/A N/A Logue JS, Morrison DK. Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy. Genes Dev Apr 1; 26(7): Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol Aug; 8(8): Carr SA, batiello SE, Ackermann BL et al. Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach. Mol Cell Proteomics Mar; 13(3): Ackermann BL. Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem Dec; 58(12): Scaffold is a trademark of Proteome Software. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. For Research use only. Not for use in diagnostic procedures. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64139-EN 0614S 6 Enrichment of /PI3K/AKT/ Proteins for Research using Immunoprecipitation and with Mass Spectrometry-based Analysis

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