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

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

Overview Purpose: Identification and quantification of EGFR/PI3K/AKT/PTEN 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. EGFR, PI3K, AKT isoforms and PTEN 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 EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 tryptic peptides. Multiple targets (EGFR, AKT isoforms, PTEN) 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 EGFR, AKT2, AKT1 and PTEN from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant EGFR 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 EGFR-PI3K-AKT signaling pathway proteins enables the precise characterization of the disease.

Methods Sample Preparation EGFR from A431 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 (EGFR, AKT isoforms, PTEN and PIK3CA) for MS applications. Multiplex IP was performed to enrich EGFR, AKT isoforms and PTEN targets simultaneously from HEK293 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.

FIGURE 1. Enrichment is necessary for medium to low abundant proteins.

Results

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

• Ab savings: All binding on outer surface

• Automation: Improves throughput and reproducibility

FIGURE 3. Evaluation of EGFR immunocapture efficiency and selectivity.

EGFR immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) EGFR 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 EGFR sequence coverage.

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 nLC-MS/MS to identify candidate quantitative peptides. Heavy isotope-labeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation.

Anti-EGFR Western

EGFR

Heavy chain

In-Solution nLC-MS/MS Results

Success Criteria: <60 >60%

P: Polyacrylamide, A: Agarose M: Magnetic

+ Anti-EGFR; - Rabbit IgG

FIGURE 4. Identification of multiple phosphorylation sites for EGFR peptides.

A

B

A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for EGFR and AKT2 peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of EGFR.

Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads

EGFR-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.

Target A431 HEK293

Anti-target Ab

Negative Control

Anti-target Ab

Negative Control

% Sequence Coverage

EGFR 65% 0% 16% 0%

AKT1 36% 2% 68% 6%

AKT2 50% 0% 82% 0%

AKT3 8% 0% 62% 0%

PTEN 16% 0% 36% 0%

PIK3CA 0% 0% 0% 0%

AKT2

EGFR

1166-Phosphoserine

693-Phosphothreonine

by PKD/PRKD1

991-Phosphoserine

451-Phosphothreonine

b₃⁺342.19

y₁₄⁺-P1448.85b₄⁺-H₂O

453.25b₁₇⁺

1826.83y₅⁺

600.44

y₁₆⁺-H₂O1754.86

y₄⁺472.50

b₁₈⁺-P1842.06

y₃⁺401.40

y₁₅⁺-P1545.87

b₁₈⁺1940.12

b₁₁⁺-P1134.60

y₁₃⁺-H₂O1415.81

b₁₁⁺1232.60

b₁₂⁺1303.60

y₁₃⁺1433.92

y₁₃²⁺-P668.54

y₁₅²⁺-P, y₁₄²⁺773.56

y₁₅⁺1643.90

y₈⁺882.59

[M+2H]²⁺-P1008.67

y₇⁺811.60

y₁₂⁺1252.78

y₁₅²⁺822.53

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\EGFR\Batch2\IP_PMS_EGFR_R1_1.raw #2257 RT: 31.37 ITMS, CID@35.00, z=+2, Mono m/z=1057.53271 Da, MH+=2114.05815 Da, Match Tol.=0.5 DaELVEPL(pT)PSGEAPNQALLR

FIGURE 5. Detection and quantitation limits of EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 peptides.

All six targets were monitored with linear quantification. EGFR, AKT2, PTEN, PIK3CA and PIK3R1 peptides were quantified from 3.9 fmol to 1000 fmol.

Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2)

EGFR Peptide 1 0.2 3.9 1000 0.9977 Peptide 2 0.2 3.9 1000 0.9997

AKT2 Peptide 1 0.2 3.9 1000 0.9998 Peptide 2 3.9 15.6 1000 0.9599

AKT1 Peptide 1 3.9 15.6 1000 0.9541

PTEN Peptide 1 0.2 3.9 1000 0.9999 Peptide 2 0.2 3.9 1000 0.9997

PIK3R1 Peptide 1 0.2 3.9 1000 0.9997 Peptide 2 0.2 3.9 1000 0.9999

PIK3CA Peptide 1 0.2 3.9 1000 0.9981

FIGURE 6. Quantitation of EGFR peptides by targeted MS.

Enrichment of EGFR from two cell lysate allowed for quantitation of two unique EGFR peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads.

Immunoprecipitation to targeted MS research application (nLC-SRM/MS)

After enrichment by IP, SRM assays enabled the quantitation of EGFR, AKT2, AKT1, PTEN proteins in the low fmol range.

FIGURE 7. Recovery of recombinant EGFR (rEGFR) from plasma matrix.

rEGFR spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more rEGFR.

rEGFR Mono Ab

Poly Ab

0 ng 0% 0%

7 ng 3% 2%

36 ng 20% 12%

180 ng 42% 26%

nLC-MS/MS (EGFR % Sequence Coverage)

nLC-SRM/MS (EGFR Peptide Quantitation)

Pierce™ Streptavidin Magnetic Beads Dynabeads® MyOne™ Streptavidin T1

A431 HEK293

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 EGFR, AKT isoforms, and PTEN in A431 and HEK293 lysates enabled detection by discovery MS and quantitation by targeted MS.

Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple isoforms and phosphorylation sites.

EGFR, AKT1, AKT2 and PTEN were quantified in the low nanogram range by nLC-SRM/MS in two cell lysates.

Enrichment of as low as 7ng recombinant EGFR in plasma matrix and 10ng of recombinant PIK3CA/PIK3R1 in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS.

Mulitplex IP to MS allowed simultaneous detection and quantification of EGFR, AKT2, AKT1 and PTEN targets.

Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein).

References 1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the

use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007 Aug; 8(8):645-54.

3. Carr SA, Abbatiello 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. 2014 Mar; 13(3):907-17.

4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620-2.

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.

FIGURE 9. Summary of EGFR-AKT pathway targets identified and quantified in two cell lines without and with enrichment.

Target Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP

EGFR A431 + + + + HEK293 - + - +

AKT1 A431 - + - + HEK293 - + - +

AKT2 A431 - + - + HEK293 - + - +

AKT3 A431 - + N/A N/A HEK293 - + N/A N/A

Grp94 A431 + + N/A N/A HEK293 + + N/A N/A

PIK3CA A431 - - N/A N/A HEK293 - - N/A N/A

PIK3R1 A431 - - N/A N/A HEK293 - - N/A N/A

PTEN A431 - + - + HEK293 - + - +

Enrichment of EGFR/PI3K/AKT/PTEN 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

FIGURE 8. Multiplex immunoprecipitation to MS research applications.

EGFR, AKT isoforms and PTEN were enriched simultaneously from HEK293 lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS.

Targets/ HEK293 lysate

% Sequence Coverage

EGFR 17%

AKT2 23%

AKT1 16%

PTEN 11%

nLC-MS/MS nLC-SRM/MS Targets/

HEK293 lysate Concentration

(fmol) Concentration

(ng)

EGFR 46 6.2

AKT2 96 5.4

AKT1 >ULOQ >ULOQ

PTEN 89 4.2

PO64139-EN 0614S

3Thermo Scientific Poster Note • PN-64139-ASMS-EN-0614S

Overview Purpose: Identification and quantification of EGFR/PI3K/AKT/PTEN 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. EGFR, PI3K, AKT isoforms and PTEN 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 EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 tryptic peptides. Multiple targets (EGFR, AKT isoforms, PTEN) 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 EGFR, AKT2, AKT1 and PTEN from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant EGFR 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 EGFR-PI3K-AKT signaling pathway proteins enables the precise characterization of the disease.

Methods Sample Preparation EGFR from A431 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 (EGFR, AKT isoforms, PTEN and PIK3CA) for MS applications. Multiplex IP was performed to enrich EGFR, AKT isoforms and PTEN targets simultaneously from HEK293 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.

FIGURE 1. Enrichment is necessary for medium to low abundant proteins.

Results

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

• Ab savings: All binding on outer surface

• Automation: Improves throughput and reproducibility

FIGURE 3. Evaluation of EGFR immunocapture efficiency and selectivity.

EGFR immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) EGFR 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 EGFR sequence coverage.

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 nLC-MS/MS to identify candidate quantitative peptides. Heavy isotope-labeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation.

Anti-EGFR Western

EGFR

Heavy chain

In-Solution nLC-MS/MS Results

Success Criteria: <60 >60%

P: Polyacrylamide, A: Agarose M: Magnetic

+ Anti-EGFR; - Rabbit IgG

FIGURE 4. Identification of multiple phosphorylation sites for EGFR peptides.

A

B

A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for EGFR and AKT2 peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of EGFR.

Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads

EGFR-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.

Target A431 HEK293

Anti-target Ab

Negative Control

Anti-target Ab

Negative Control

% Sequence Coverage

EGFR 65% 0% 16% 0%

AKT1 36% 2% 68% 6%

AKT2 50% 0% 82% 0%

AKT3 8% 0% 62% 0%

PTEN 16% 0% 36% 0%

PIK3CA 0% 0% 0% 0%

AKT2

EGFR

1166-Phosphoserine

693-Phosphothreonine

by PKD/PRKD1

991-Phosphoserine

451-Phosphothreonine

b₃⁺342.19

y₁₄⁺-P1448.85b₄⁺-H₂O

453.25b₁₇⁺

1826.83y₅⁺

600.44

y₁₆⁺-H₂O1754.86

y₄⁺472.50

b₁₈⁺-P1842.06

y₃⁺401.40

y₁₅⁺-P1545.87

b₁₈⁺1940.12

b₁₁⁺-P1134.60

y₁₃⁺-H₂O1415.81

b₁₁⁺1232.60

b₁₂⁺1303.60

y₁₃⁺1433.92

y₁₃²⁺-P668.54

y₁₅²⁺-P, y₁₄²⁺773.56

y₁₅⁺1643.90

y₈⁺882.59

[M+2H]²⁺-P1008.67

y₇⁺811.60

y₁₂⁺1252.78

y₁₅²⁺822.53

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\EGFR\Batch2\IP_PMS_EGFR_R1_1.raw #2257 RT: 31.37 ITMS, CID@35.00, z=+2, Mono m/z=1057.53271 Da, MH+=2114.05815 Da, Match Tol.=0.5 DaELVEPL(pT)PSGEAPNQALLR

FIGURE 5. Detection and quantitation limits of EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 peptides.

All six targets were monitored with linear quantification. EGFR, AKT2, PTEN, PIK3CA and PIK3R1 peptides were quantified from 3.9 fmol to 1000 fmol.

Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2)

EGFR Peptide 1 0.2 3.9 1000 0.9977 Peptide 2 0.2 3.9 1000 0.9997

AKT2 Peptide 1 0.2 3.9 1000 0.9998 Peptide 2 3.9 15.6 1000 0.9599

AKT1 Peptide 1 3.9 15.6 1000 0.9541

PTEN Peptide 1 0.2 3.9 1000 0.9999 Peptide 2 0.2 3.9 1000 0.9997

PIK3R1 Peptide 1 0.2 3.9 1000 0.9997 Peptide 2 0.2 3.9 1000 0.9999

PIK3CA Peptide 1 0.2 3.9 1000 0.9981

FIGURE 6. Quantitation of EGFR peptides by targeted MS.

Enrichment of EGFR from two cell lysate allowed for quantitation of two unique EGFR peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads.

Immunoprecipitation to targeted MS research application (nLC-SRM/MS)

After enrichment by IP, SRM assays enabled the quantitation of EGFR, AKT2, AKT1, PTEN proteins in the low fmol range.

FIGURE 7. Recovery of recombinant EGFR (rEGFR) from plasma matrix.

rEGFR spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more rEGFR.

rEGFR Mono Ab

Poly Ab

0 ng 0% 0%

7 ng 3% 2%

36 ng 20% 12%

180 ng 42% 26%

nLC-MS/MS (EGFR % Sequence Coverage)

nLC-SRM/MS (EGFR Peptide Quantitation)

Pierce™ Streptavidin Magnetic Beads Dynabeads® MyOne™ Streptavidin T1

A431 HEK293

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 EGFR, AKT isoforms, and PTEN in A431 and HEK293 lysates enabled detection by discovery MS and quantitation by targeted MS.

Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple isoforms and phosphorylation sites.

EGFR, AKT1, AKT2 and PTEN were quantified in the low nanogram range by nLC-SRM/MS in two cell lysates.

Enrichment of as low as 7ng recombinant EGFR in plasma matrix and 10ng of recombinant PIK3CA/PIK3R1 in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS.

Mulitplex IP to MS allowed simultaneous detection and quantification of EGFR, AKT2, AKT1 and PTEN targets.

Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein).

References 1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the

use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007 Aug; 8(8):645-54.

3. Carr SA, Abbatiello 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. 2014 Mar; 13(3):907-17.

4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620-2.

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.

FIGURE 9. Summary of EGFR-AKT pathway targets identified and quantified in two cell lines without and with enrichment.

Target Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP

EGFR A431 + + + + HEK293 - + - +

AKT1 A431 - + - + HEK293 - + - +

AKT2 A431 - + - + HEK293 - + - +

AKT3 A431 - + N/A N/A HEK293 - + N/A N/A

Grp94 A431 + + N/A N/A HEK293 + + N/A N/A

PIK3CA A431 - - N/A N/A HEK293 - - N/A N/A

PIK3R1 A431 - - N/A N/A HEK293 - - N/A N/A

PTEN A431 - + - + HEK293 - + - +

Enrichment of EGFR/PI3K/AKT/PTEN 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

FIGURE 8. Multiplex immunoprecipitation to MS research applications.

EGFR, AKT isoforms and PTEN were enriched simultaneously from HEK293 lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS.

Targets/ HEK293 lysate

% Sequence Coverage

EGFR 17%

AKT2 23%

AKT1 16%

PTEN 11%

nLC-MS/MS nLC-SRM/MS Targets/

HEK293 lysate Concentration

(fmol) Concentration

(ng)

EGFR 46 6.2

AKT2 96 5.4

AKT1 >ULOQ >ULOQ

PTEN 89 4.2

PO64139-EN 0614S

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

Overview Purpose: Identification and quantification of EGFR/PI3K/AKT/PTEN 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. EGFR, PI3K, AKT isoforms and PTEN 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 EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 tryptic peptides. Multiple targets (EGFR, AKT isoforms, PTEN) 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 EGFR, AKT2, AKT1 and PTEN from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant EGFR 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 EGFR-PI3K-AKT signaling pathway proteins enables the precise characterization of the disease.

Methods Sample Preparation EGFR from A431 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 (EGFR, AKT isoforms, PTEN and PIK3CA) for MS applications. Multiplex IP was performed to enrich EGFR, AKT isoforms and PTEN targets simultaneously from HEK293 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.

FIGURE 1. Enrichment is necessary for medium to low abundant proteins.

Results

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

• Ab savings: All binding on outer surface

• Automation: Improves throughput and reproducibility

FIGURE 3. Evaluation of EGFR immunocapture efficiency and selectivity.

EGFR immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) EGFR 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 EGFR sequence coverage.

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 nLC-MS/MS to identify candidate quantitative peptides. Heavy isotope-labeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation.

Anti-EGFR Western

EGFR

Heavy chain

In-Solution nLC-MS/MS Results

Success Criteria: <60 >60%

P: Polyacrylamide, A: Agarose M: Magnetic

+ Anti-EGFR; - Rabbit IgG

FIGURE 4. Identification of multiple phosphorylation sites for EGFR peptides.

A

B

A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for EGFR and AKT2 peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of EGFR.

Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads

EGFR-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.

Target A431 HEK293

Anti-target Ab

Negative Control

Anti-target Ab

Negative Control

% Sequence Coverage

EGFR 65% 0% 16% 0%

AKT1 36% 2% 68% 6%

AKT2 50% 0% 82% 0%

AKT3 8% 0% 62% 0%

PTEN 16% 0% 36% 0%

PIK3CA 0% 0% 0% 0%

AKT2

EGFR

1166-Phosphoserine

693-Phosphothreonine

by PKD/PRKD1

991-Phosphoserine

451-Phosphothreonine

b₃⁺342.19

y₁₄⁺-P1448.85b₄⁺-H₂O

453.25b₁₇⁺

1826.83y₅⁺

600.44

y₁₆⁺-H₂O1754.86

y₄⁺472.50

b₁₈⁺-P1842.06

y₃⁺401.40

y₁₅⁺-P1545.87

b₁₈⁺1940.12

b₁₁⁺-P1134.60

y₁₃⁺-H₂O1415.81

b₁₁⁺1232.60

b₁₂⁺1303.60

y₁₃⁺1433.92

y₁₃²⁺-P668.54

y₁₅²⁺-P, y₁₄²⁺773.56

y₁₅⁺1643.90

y₈⁺882.59

[M+2H]²⁺-P1008.67

y₇⁺811.60

y₁₂⁺1252.78

y₁₅²⁺822.53

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\EGFR\Batch2\IP_PMS_EGFR_R1_1.raw #2257 RT: 31.37 ITMS, CID@35.00, z=+2, Mono m/z=1057.53271 Da, MH+=2114.05815 Da, Match Tol.=0.5 DaELVEPL(pT)PSGEAPNQALLR

FIGURE 5. Detection and quantitation limits of EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 peptides.

All six targets were monitored with linear quantification. EGFR, AKT2, PTEN, PIK3CA and PIK3R1 peptides were quantified from 3.9 fmol to 1000 fmol.

Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2)

EGFR Peptide 1 0.2 3.9 1000 0.9977 Peptide 2 0.2 3.9 1000 0.9997

AKT2 Peptide 1 0.2 3.9 1000 0.9998 Peptide 2 3.9 15.6 1000 0.9599

AKT1 Peptide 1 3.9 15.6 1000 0.9541

PTEN Peptide 1 0.2 3.9 1000 0.9999 Peptide 2 0.2 3.9 1000 0.9997

PIK3R1 Peptide 1 0.2 3.9 1000 0.9997 Peptide 2 0.2 3.9 1000 0.9999

PIK3CA Peptide 1 0.2 3.9 1000 0.9981

FIGURE 6. Quantitation of EGFR peptides by targeted MS.

Enrichment of EGFR from two cell lysate allowed for quantitation of two unique EGFR peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads.

Immunoprecipitation to targeted MS research application (nLC-SRM/MS)

After enrichment by IP, SRM assays enabled the quantitation of EGFR, AKT2, AKT1, PTEN proteins in the low fmol range.

FIGURE 7. Recovery of recombinant EGFR (rEGFR) from plasma matrix.

rEGFR spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more rEGFR.

rEGFR Mono Ab

Poly Ab

0 ng 0% 0%

7 ng 3% 2%

36 ng 20% 12%

180 ng 42% 26%

nLC-MS/MS (EGFR % Sequence Coverage)

nLC-SRM/MS (EGFR Peptide Quantitation)

Pierce™ Streptavidin Magnetic Beads Dynabeads® MyOne™ Streptavidin T1

A431 HEK293

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 EGFR, AKT isoforms, and PTEN in A431 and HEK293 lysates enabled detection by discovery MS and quantitation by targeted MS.

Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple isoforms and phosphorylation sites.

EGFR, AKT1, AKT2 and PTEN were quantified in the low nanogram range by nLC-SRM/MS in two cell lysates.

Enrichment of as low as 7ng recombinant EGFR in plasma matrix and 10ng of recombinant PIK3CA/PIK3R1 in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS.

Mulitplex IP to MS allowed simultaneous detection and quantification of EGFR, AKT2, AKT1 and PTEN targets.

Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein).

References 1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the

use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007 Aug; 8(8):645-54.

3. Carr SA, Abbatiello 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. 2014 Mar; 13(3):907-17.

4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620-2.

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.

FIGURE 9. Summary of EGFR-AKT pathway targets identified and quantified in two cell lines without and with enrichment.

Target Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP

EGFR A431 + + + + HEK293 - + - +

AKT1 A431 - + - + HEK293 - + - +

AKT2 A431 - + - + HEK293 - + - +

AKT3 A431 - + N/A N/A HEK293 - + N/A N/A

Grp94 A431 + + N/A N/A HEK293 + + N/A N/A

PIK3CA A431 - - N/A N/A HEK293 - - N/A N/A

PIK3R1 A431 - - N/A N/A HEK293 - - N/A N/A

PTEN A431 - + - + HEK293 - + - +

Enrichment of EGFR/PI3K/AKT/PTEN 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

FIGURE 8. Multiplex immunoprecipitation to MS research applications.

EGFR, AKT isoforms and PTEN were enriched simultaneously from HEK293 lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS.

Targets/ HEK293 lysate

% Sequence Coverage

EGFR 17%

AKT2 23%

AKT1 16%

PTEN 11%

nLC-MS/MS nLC-SRM/MS Targets/

HEK293 lysate Concentration

(fmol) Concentration

(ng)

EGFR 46 6.2

AKT2 96 5.4

AKT1 >ULOQ >ULOQ

PTEN 89 4.2

PO64139-EN 0614S

5Thermo Scientific Poster Note • PN-64139-ASMS-EN-0614S

Overview Purpose: Identification and quantification of EGFR/PI3K/AKT/PTEN 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. EGFR, PI3K, AKT isoforms and PTEN 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 EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 tryptic peptides. Multiple targets (EGFR, AKT isoforms, PTEN) 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 EGFR, AKT2, AKT1 and PTEN from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant EGFR 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 EGFR-PI3K-AKT signaling pathway proteins enables the precise characterization of the disease.

Methods Sample Preparation EGFR from A431 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 (EGFR, AKT isoforms, PTEN and PIK3CA) for MS applications. Multiplex IP was performed to enrich EGFR, AKT isoforms and PTEN targets simultaneously from HEK293 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.

FIGURE 1. Enrichment is necessary for medium to low abundant proteins.

Results

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

• Ab savings: All binding on outer surface

• Automation: Improves throughput and reproducibility

FIGURE 3. Evaluation of EGFR immunocapture efficiency and selectivity.

EGFR immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) EGFR 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 EGFR sequence coverage.

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 nLC-MS/MS to identify candidate quantitative peptides. Heavy isotope-labeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation.

Anti-EGFR Western

EGFR

Heavy chain

In-Solution nLC-MS/MS Results

Success Criteria: <60 >60%

P: Polyacrylamide, A: Agarose M: Magnetic

+ Anti-EGFR; - Rabbit IgG

FIGURE 4. Identification of multiple phosphorylation sites for EGFR peptides.

A

B

A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for EGFR and AKT2 peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of EGFR.

Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads

EGFR-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.

Target A431 HEK293

Anti-target Ab

Negative Control

Anti-target Ab

Negative Control

% Sequence Coverage

EGFR 65% 0% 16% 0%

AKT1 36% 2% 68% 6%

AKT2 50% 0% 82% 0%

AKT3 8% 0% 62% 0%

PTEN 16% 0% 36% 0%

PIK3CA 0% 0% 0% 0%

AKT2

EGFR

1166-Phosphoserine

693-Phosphothreonine

by PKD/PRKD1

991-Phosphoserine

451-Phosphothreonine

b₃⁺342.19

y₁₄⁺-P1448.85b₄⁺-H₂O

453.25b₁₇⁺

1826.83y₅⁺

600.44

y₁₆⁺-H₂O1754.86

y₄⁺472.50

b₁₈⁺-P1842.06

y₃⁺401.40

y₁₅⁺-P1545.87

b₁₈⁺1940.12

b₁₁⁺-P1134.60

y₁₃⁺-H₂O1415.81

b₁₁⁺1232.60

b₁₂⁺1303.60

y₁₃⁺1433.92

y₁₃²⁺-P668.54

y₁₅²⁺-P, y₁₄²⁺773.56

y₁₅⁺1643.90

y₈⁺882.59

[M+2H]²⁺-P1008.67

y₇⁺811.60

y₁₂⁺1252.78

y₁₅²⁺822.53

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\EGFR\Batch2\IP_PMS_EGFR_R1_1.raw #2257 RT: 31.37 ITMS, CID@35.00, z=+2, Mono m/z=1057.53271 Da, MH+=2114.05815 Da, Match Tol.=0.5 DaELVEPL(pT)PSGEAPNQALLR

FIGURE 5. Detection and quantitation limits of EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 peptides.

All six targets were monitored with linear quantification. EGFR, AKT2, PTEN, PIK3CA and PIK3R1 peptides were quantified from 3.9 fmol to 1000 fmol.

Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2)

EGFR Peptide 1 0.2 3.9 1000 0.9977 Peptide 2 0.2 3.9 1000 0.9997

AKT2 Peptide 1 0.2 3.9 1000 0.9998 Peptide 2 3.9 15.6 1000 0.9599

AKT1 Peptide 1 3.9 15.6 1000 0.9541

PTEN Peptide 1 0.2 3.9 1000 0.9999 Peptide 2 0.2 3.9 1000 0.9997

PIK3R1 Peptide 1 0.2 3.9 1000 0.9997 Peptide 2 0.2 3.9 1000 0.9999

PIK3CA Peptide 1 0.2 3.9 1000 0.9981

FIGURE 6. Quantitation of EGFR peptides by targeted MS.

Enrichment of EGFR from two cell lysate allowed for quantitation of two unique EGFR peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads.

Immunoprecipitation to targeted MS research application (nLC-SRM/MS)

After enrichment by IP, SRM assays enabled the quantitation of EGFR, AKT2, AKT1, PTEN proteins in the low fmol range.

FIGURE 7. Recovery of recombinant EGFR (rEGFR) from plasma matrix.

rEGFR spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more rEGFR.

rEGFR Mono Ab

Poly Ab

0 ng 0% 0%

7 ng 3% 2%

36 ng 20% 12%

180 ng 42% 26%

nLC-MS/MS (EGFR % Sequence Coverage)

nLC-SRM/MS (EGFR Peptide Quantitation)

Pierce™ Streptavidin Magnetic Beads Dynabeads® MyOne™ Streptavidin T1

A431 HEK293

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 EGFR, AKT isoforms, and PTEN in A431 and HEK293 lysates enabled detection by discovery MS and quantitation by targeted MS.

Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple isoforms and phosphorylation sites.

EGFR, AKT1, AKT2 and PTEN were quantified in the low nanogram range by nLC-SRM/MS in two cell lysates.

Enrichment of as low as 7ng recombinant EGFR in plasma matrix and 10ng of recombinant PIK3CA/PIK3R1 in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS.

Mulitplex IP to MS allowed simultaneous detection and quantification of EGFR, AKT2, AKT1 and PTEN targets.

Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein).

References 1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the

use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007 Aug; 8(8):645-54.

3. Carr SA, Abbatiello 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. 2014 Mar; 13(3):907-17.

4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620-2.

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.

FIGURE 9. Summary of EGFR-AKT pathway targets identified and quantified in two cell lines without and with enrichment.

Target Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP

EGFR A431 + + + + HEK293 - + - +

AKT1 A431 - + - + HEK293 - + - +

AKT2 A431 - + - + HEK293 - + - +

AKT3 A431 - + N/A N/A HEK293 - + N/A N/A

Grp94 A431 + + N/A N/A HEK293 + + N/A N/A

PIK3CA A431 - - N/A N/A HEK293 - - N/A N/A

PIK3R1 A431 - - N/A N/A HEK293 - - N/A N/A

PTEN A431 - + - + HEK293 - + - +

Enrichment of EGFR/PI3K/AKT/PTEN 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

FIGURE 8. Multiplex immunoprecipitation to MS research applications.

EGFR, AKT isoforms and PTEN were enriched simultaneously from HEK293 lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS.

Targets/ HEK293 lysate

% Sequence Coverage

EGFR 17%

AKT2 23%

AKT1 16%

PTEN 11%

nLC-MS/MS nLC-SRM/MS Targets/

HEK293 lysate Concentration

(fmol) Concentration

(ng)

EGFR 46 6.2

AKT2 96 5.4

AKT1 >ULOQ >ULOQ

PTEN 89 4.2

PO64139-EN 0614S

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

Overview Purpose: Identification and quantification of EGFR/PI3K/AKT/PTEN 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. EGFR, PI3K, AKT isoforms and PTEN 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 EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 tryptic peptides. Multiple targets (EGFR, AKT isoforms, PTEN) 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 EGFR, AKT2, AKT1 and PTEN from two cell lysates enabled MS detection and quantitation. Enrichment of as low as 7ng recombinant EGFR 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 EGFR-PI3K-AKT signaling pathway proteins enables the precise characterization of the disease.

Methods Sample Preparation EGFR from A431 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 (EGFR, AKT isoforms, PTEN and PIK3CA) for MS applications. Multiplex IP was performed to enrich EGFR, AKT isoforms and PTEN targets simultaneously from HEK293 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.

FIGURE 1. Enrichment is necessary for medium to low abundant proteins.

Results

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

• Ab savings: All binding on outer surface

• Automation: Improves throughput and reproducibility

FIGURE 3. Evaluation of EGFR immunocapture efficiency and selectivity.

EGFR immunoprecipitation was used to evaluate directly coupled antibody or biotinylated antibody with immobilized streptavidin resin. A) Capture efficiency was determined by Western blot. B) EGFR 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 EGFR sequence coverage.

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 nLC-MS/MS to identify candidate quantitative peptides. Heavy isotope-labeled, quantitative peptide standards are then used in targeted SRM or MRM research methods for absolute quantitation.

Anti-EGFR Western

EGFR

Heavy chain

In-Solution nLC-MS/MS Results

Success Criteria: <60 >60%

P: Polyacrylamide, A: Agarose M: Magnetic

+ Anti-EGFR; - Rabbit IgG

FIGURE 4. Identification of multiple phosphorylation sites for EGFR peptides.

A

B

A) IP-MS allowed simultaneous analysis of multiple phosphorylation sites for EGFR and AKT2 peptides. B) MS/MS spectra of ELVEPL(pT)PSGEAPNQALLR peptide showing phosphothreonine residue at T693 of EGFR.

Enrichment of medium to low abundant targets using Thermo Scientific Pierce Streptavidin Coated Magnetic Beads

EGFR-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.

Target A431 HEK293

Anti-target Ab

Negative Control

Anti-target Ab

Negative Control

% Sequence Coverage

EGFR 65% 0% 16% 0%

AKT1 36% 2% 68% 6%

AKT2 50% 0% 82% 0%

AKT3 8% 0% 62% 0%

PTEN 16% 0% 36% 0%

PIK3CA 0% 0% 0% 0%

AKT2

EGFR

1166-Phosphoserine

693-Phosphothreonine

by PKD/PRKD1

991-Phosphoserine

451-Phosphothreonine

b₃⁺342.19

y₁₄⁺-P1448.85b₄⁺-H₂O

453.25b₁₇⁺

1826.83y₅⁺

600.44

y₁₆⁺-H₂O1754.86

y₄⁺472.50

b₁₈⁺-P1842.06

y₃⁺401.40

y₁₅⁺-P1545.87

b₁₈⁺1940.12

b₁₁⁺-P1134.60

y₁₃⁺-H₂O1415.81

b₁₁⁺1232.60

b₁₂⁺1303.60

y₁₃⁺1433.92

y₁₃²⁺-P668.54

y₁₅²⁺-P, y₁₄²⁺773.56

y₁₅⁺1643.90

y₈⁺882.59

[M+2H]²⁺-P1008.67

y₇⁺811.60

y₁₂⁺1252.78

y₁₅²⁺822.53

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: R:\Bhavin\IP-MS\IPMS_5Kits_May2013\EGFR\Batch2\IP_PMS_EGFR_R1_1.raw #2257 RT: 31.37 ITMS, CID@35.00, z=+2, Mono m/z=1057.53271 Da, MH+=2114.05815 Da, Match Tol.=0.5 DaELVEPL(pT)PSGEAPNQALLR

FIGURE 5. Detection and quantitation limits of EGFR, AKT2, AKT1, PTEN, PIK3CA and PIK3R1 peptides.

All six targets were monitored with linear quantification. EGFR, AKT2, PTEN, PIK3CA and PIK3R1 peptides were quantified from 3.9 fmol to 1000 fmol.

Target Peptide No. LOD (fmol) LLOQ (fmol) ULOQ (fmol) Linearity (R2)

EGFR Peptide 1 0.2 3.9 1000 0.9977 Peptide 2 0.2 3.9 1000 0.9997

AKT2 Peptide 1 0.2 3.9 1000 0.9998 Peptide 2 3.9 15.6 1000 0.9599

AKT1 Peptide 1 3.9 15.6 1000 0.9541

PTEN Peptide 1 0.2 3.9 1000 0.9999 Peptide 2 0.2 3.9 1000 0.9997

PIK3R1 Peptide 1 0.2 3.9 1000 0.9997 Peptide 2 0.2 3.9 1000 0.9999

PIK3CA Peptide 1 0.2 3.9 1000 0.9981

FIGURE 6. Quantitation of EGFR peptides by targeted MS.

Enrichment of EGFR from two cell lysate allowed for quantitation of two unique EGFR peptides by targeted MS. Better recovery was observed with Pierce Streptavidin (SA) Magnetic beads compared to Dynabeads MyOne Streptavidin T1 beads.

Immunoprecipitation to targeted MS research application (nLC-SRM/MS)

After enrichment by IP, SRM assays enabled the quantitation of EGFR, AKT2, AKT1, PTEN proteins in the low fmol range.

FIGURE 7. Recovery of recombinant EGFR (rEGFR) from plasma matrix.

rEGFR spiked into 1mg plasma is detected and quantitated at >7ng (52 fmol). Monoclonal antibody recovered more rEGFR.

rEGFR Mono Ab

Poly Ab

0 ng 0% 0%

7 ng 3% 2%

36 ng 20% 12%

180 ng 42% 26%

nLC-MS/MS (EGFR % Sequence Coverage)

nLC-SRM/MS (EGFR Peptide Quantitation)

Pierce™ Streptavidin Magnetic Beads Dynabeads® MyOne™ Streptavidin T1

A431 HEK293

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 EGFR, AKT isoforms, and PTEN in A431 and HEK293 lysates enabled detection by discovery MS and quantitation by targeted MS.

Immunoprecipitation of EGFR and AKT2 resulted in simultaneous analysis of multiple isoforms and phosphorylation sites.

EGFR, AKT1, AKT2 and PTEN were quantified in the low nanogram range by nLC-SRM/MS in two cell lysates.

Enrichment of as low as 7ng recombinant EGFR in plasma matrix and 10ng of recombinant PIK3CA/PIK3R1 in cell lysate (data not shown) enabled absolute quantitation by targeted SRM-MS.

Mulitplex IP to MS allowed simultaneous detection and quantification of EGFR, AKT2, AKT1 and PTEN targets.

Future work will focus on optimization of IP conditions to enrich lower abundant targets (<1ng total protein).

References 1. Logue JS, Morrison DK. Complexity in the signaling network: insights from the

use of targeted inhibitors in cancer therapy. Genes Dev. 2012 Apr 1; 26(7):641-50.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007 Aug; 8(8):645-54.

3. Carr SA, Abbatiello 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. 2014 Mar; 13(3):907-17.

4. Ackermann BL . Understanding the role of immunoaffinity-based mass spectrometry methods for clinical applications. Clin Chem. 2012 Dec; 58(12):1620-2.

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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.

FIGURE 9. Summary of EGFR-AKT pathway targets identified and quantified in two cell lines without and with enrichment.

Target Cell line Detected by Orbitrap Quantified by SRM Neat Enriched-IP Neat Enriched-IP

EGFR A431 + + + + HEK293 - + - +

AKT1 A431 - + - + HEK293 - + - +

AKT2 A431 - + - + HEK293 - + - +

AKT3 A431 - + N/A N/A HEK293 - + N/A N/A

Grp94 A431 + + N/A N/A HEK293 + + N/A N/A

PIK3CA A431 - - N/A N/A HEK293 - - N/A N/A

PIK3R1 A431 - - N/A N/A HEK293 - - N/A N/A

PTEN A431 - + - + HEK293 - + - +

Enrichment of EGFR/PI3K/AKT/PTEN 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

FIGURE 8. Multiplex immunoprecipitation to MS research applications.

EGFR, AKT isoforms and PTEN were enriched simultaneously from HEK293 lysate with biotinylated antibodies, captured with Pierce Streptavidin coated magnetic beads. All four targets were identified and quantified by MS.

Targets/ HEK293 lysate

% Sequence Coverage

EGFR 17%

AKT2 23%

AKT1 16%

PTEN 11%

nLC-MS/MS nLC-SRM/MS Targets/

HEK293 lysate Concentration

(fmol) Concentration

(ng)

EGFR 46 6.2

AKT2 96 5.4

AKT1 >ULOQ >ULOQ

PTEN 89 4.2

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