A High-Throughput, Multiplexed Microfluidic Method Utilizing An Optically Barcoded Drop Library
High-throughput Multiplexed SISCAPA Assay for Targeted ...€¦ · High-throughput Multiplexed...
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Covance is the drug development business of Laboratory Corporation of America ® Holdings (LabCorp ® ). Content of this material was developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance. High-throughput Multiplexed SISCAPA ® Assay for Targeted Protein Quantitation by LC-MS/MS 1 Bob Xiong, 2 Randall Brock, 1 Lori Wright, 1 Lynn Garcowski, 1 Monica Germann, 1 Brian Nofsinger and 1 Mike Allen 1 Tandem Labs, Laboratory Corporation of America ® Holdings 2 Onconome, Inc., Redmond, WA Introduction Material and Method Material and Method (continued) Results and Discussion SISCAPA ® (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) assays are a popular tool for validation of candidate protein biomarkers. They are used in research and in the clinic as a highly-specific and high-throughput alternative to immunoassay and traditional mass spectrometry. We report here an implementation of 96-well protein digestion and Conclusion References As a sample enrichment technique, SISCAPA was successfully incorporated in a 11-plex LC-MRM assay to determine the abundance of 11 proteins in human serum. The 96-well format digestion on a PCR thermal cycler and automated liquid handling workstation allows for high-throughput quantitative analyses of multiple target proteins in biological samples by LC- MRM. In a multiplexing SISCAPA-LC-MRM assay, optimization of the coupling of magnetic beads with monoclonal antibodies is critical for a successful outcome of sample enrichment and subsequent LC-MRM detection. Figure 1 High-throughput multiplexed SISCAPA-LC-MRM workflow Table 1 Peptide fragment transitions monitored in the 11-plex MRM Table 2 Figure 2 Representative chromatograms for Peptide 3 Figure 3 Representative chromatograms for Peptide SISCAPA enrichment produced clean background (see Figures 2 and 3 for representative chromatograms). As a result, the signal-to- noise ratio was much improved compared to extractions such as protein precipitation and solid-phase extraction (data not shown). Improved signal-to-noise ratios translate into lower levels of detection/ quantitation thus yielding higher sensitivity of a LC-MRM assay. In the present study, lower limit of quantitation (LLOQ) was achieved below 1 ng/mL for the majority of the 11 analytes (data not shown). In the absence of protein standards, we used blank matrix samples to calculate the variability of endogenous tryptic peptides across four runs. The coefficient of variation (CV) varied significantly both within and between runs depending on the analytes (Table 2). In general, weaker signals displayed greater CVs within runs (e.g. Peptides 3, 8, 10, 11 and 13 as shown in Table 2). We observed greater inter-assay CVs for Peptides 4 (34%) and 12 (40%) even though the intra-assay CVs were 7 – 12% and 9 – 13%, respectively. The greater inter-assay CVs for Peptides 4 and 12 were probably a reflection of the varied digestion efficiency and/or extraction between runs, which highlights the importance of including protein QC samples in MRM assays, whenever possible, so the variation in digestion efficiency/extraction can be addressed. We compared the coupling of magnetic beads with the mAbs in a cocktail mixture and individual mAbs. Two of the 11 mAbs (SAT-71 and SAT-78) failed to produce signals following the cocktail coupling, while the individually coupled bead-mAb complexes produced signals for all the 11 peptides (data not shown). We suspect that SAT-71 and SAT- 78 were out-competed by others in the cocktail coupling experiment. In the present study, we performed the individual bead-mAb coupling and combined the individually coupled bead-mAb complexes prior to use to ensure that all mAbs were coupled to beads and all target peptides were pulled down during sample enrichment. SISCAPA mediated sample enrichment for 11-plex targeted protein quantification by LC-MS/MS. The results are encouraging with respect to the reduction of background complexity leading to improved signal-to-noise ratio and sensitivity, albeit rather variable in reproducibility across multiple runs, depending on the analytes. Under the direction of SISCAPA Assay Technologies, Inc. (SAT; Washington, DC), synthetic proteotypic peptide surrogates from each biomarker protein were synthesized, coupled to keyhole limpet hemocyanin as carrier, and the conjugates were used to immunize rabbits for derivation of rabbit monoclonal antibodies (RabMAbs) by Epitomics, Inc. (Burlingame, CA). To select hybridomas secreting anti-peptide antibodies, hybridoma supernatants from each fusion were tested by SAT in peptide ELISA (Razavi et al., 2011). Positive rabbit hybridoma supernatants were subsequently screened for high affinity antibodies using a combination of immunoscreening-mass spectrometry (Razavi et al., 2011) and surface plasmon resonance (Pope et al., 2009) assays under conditions that measure monovalent interactions (reflecting antibody affinity rather than avidity). Antibodies that were able to bind a target peptide from solution phase and retain it during washing for a minimum of 10 minutes were identified, as required for use in high-sensitivity SISCAPA assays. The selected antibodies were produced in milligram quantities by recombinant expression at Epitomics and kinetic analysis of antibody-peptide binding was subsequently performed by surface plasmon resonance at SAT. All of the peptide-specific RabMAb reagents exhibited high affinity with low dissociation constants. Finally, the recombinant RabMAbs were characterized in multiplex SISCAPA assays using human plasma digest matrix, and the amount of each antibody was optimized for use in peptide capture. Anti-peptide monoclonal antibodies Dynabeads and individual mAb coupling I. Prepare Dynabeads ® (Dynal, Inc.) a. Resuspend beads by vortexing vial > 30sec while tilting and rotating vial until no material is settled on the bottom of the container. b. Transfer 600μL (1.5mg) Dynabeads to a 2mL polypropylene (PPE) tube. c. Place the tube on the magnet to separate the beads from solution and remove the supernatant. d. Remove the tube from the magnet. II. Wash Beads (2X) a. Add 1000μL of 1xPBS+0.03%CHAPS to the tube, cap and vortex gently. b. Place the tube on the magnet to separate the beads from solution and remove the supernatant. c. Repeat wash step (II.a and II.b). d. Transfer washed beads from the 2mL polypropylene (PPE) tube to a 15mL polypropylene (PPE) conical tube. III. Bind mAb (individual mAb coupling) a. Add 240μL mAb (in 1xPBS+0.03%CHAPS) to the 15mL tube containing the beads. b. Add an additional 1.92 mL 1xPBS+0.03%CHAPS to the 15mL tube containing beads to dilute to a final volume of about 6mL including mAb volumes, cap and wrap capped tube with parafilm to prevent leakage. c. Incubate at room temperature on rotational shaker for 1 hour. IV. Bead-mAb complex Storage a. Store bead-mAb complex in the 15mL tube at 4°C until needed. Sample tryptic digestion (performed on GeneAmp ® PCR System 9700 (Roche Molecular Systems, Inc.)) 1. Sample volume: 135 μL (containing 4 μL of human serum, internal standard peptides, appropriate amount of calibrator peptides, in 67.5 mM ammonium bicarbonate) 2. Denaturation: 95 o C, 10 min. 3. Quenching: 4 o C, 10 min. 4. Reduction: 13 mM DTT, 50 o C, 30 min. 5. Alkylation: 23 mM Iodoacetamide, 25 o C, 30 min. 6. Digestion: 5 μg Trypsin (enzyme:substrate = 1:60, w/w), 37 o C, 12-16 hours Sample enrichment (performed on Hamilton Microlab ® Star liquid handling workstation) 1. Combine the 11 individually coupled bead-mAb complexes to create bead-mAb cocktail. 2. Manually add 100μL bead-mAb cocktail to wells in 2mL 96-well NUNC plate using a multi channel pipette. 3. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the supernatant. 4. Add entire digested sample (~135μL) from PCR plate to the 2mL 96-well NUNC plate containing washed beads. 5. Add 500μL of 1XPBS+0.03%CHAPS to each well, cap plate and incubate for 1 hour at room temp using the Vibrax VXR Basic at 900-1000rpm. 6. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the solvent. 7. Wash wells containing beads with 600μL of 1xPBS+0.03%CHAPS. 8. Shake using robotic shaker for 2 minutes at 1000 rpm. 9. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the wash solvent. 10. Wash wells containing beads with 600μL of 1xPBS+0.03%CHAPS a second time. 11. Shake using Eva shaker for 2 minutes at 1000 rpm. 12. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the wash solvent. 13. Add 125μL of 0.1% formic acid in water + 0.03% CHAPS to each well for peptide elution. 14. Cap plate and place on Eva shaker for 5 minutes at 1000 rpm. 15. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution. 16. Remove the elution solvent and transfer into a 1mL 96-well NUNC plate and cap for analysis. Liquid chromatography System: Shimadzu CBM-20A, LC-20AD Column: Agilent AdvanceBio ® Peptide Map 2.1 x 100mm 2.7-micron 600Bar Column Temp: 40 o C MPA: water with 0.1% formic acid MPB: acetonitrile with 0.1% formic acid Wash 1: 60/30/10 acetonitrile/isopropanol/acetone Wash 2: 75/25 water/acetonitrile with 1% formic acid Total Flow: 0.3 mL/min Gradient: 0.8 min 0-20%B, 0.8 min 20-40%B, 0.4 min 40%B, 0.2 min 40-65%B, 0.6 min 65-95%B, 0.7 min 95%B, 0.5 min 95-0%B, 1 min 0%B (total 5 min.) Mass spectrometry Instrument: AB Sciex ® API 5000 Polarity: Positive Scan Type: MRM Ion Source: Turbo Spray IS: 1500 TEM: 500 DP: 100 CEM: 2500 Table 2 Intra- and Inter-assay variability across four runs. Variability was based on the peak area ratios of 11 endogenous tryptic peptides in blank matrix samples (13 blank matrix samples per run). The intra- and inter-assay CV (%) was calculated for each peptide. Morteza Razavi, Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson. MALDI Immunoscreening (MiSCREEN): A Method for Selection of Anti-peptide Monoclonal Antibodies for Use in Immunoproteomics. Journal of Immunological Methods (2011); 364:50-64. Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson. Anti-peptide antibody screening: selection of high affinity monoclonal reagents by a refined surface plasmon resonance technique. Journal of Immunological Methods (2009); 341:86-96.
Transcript of High-throughput Multiplexed SISCAPA Assay for Targeted ...€¦ · High-throughput Multiplexed...
Covance is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Content of this material was developed by scientists who at the time were affiliated with LabCorp Clinical Trials or Tandem Labs, now part of Covance.
High-throughput Multiplexed SISCAPA® Assay for Targeted Protein Quantitation by LC-MS/MS1Bob Xiong, 2Randall Brock, 1Lori Wright, 1Lynn Garcowski, 1Monica Germann, 1Brian Nofsinger and 1Mike Allen1Tandem Labs, Laboratory Corporation of America® Holdings2Onconome, Inc., Redmond, WA
Introduction
Material and Method
Material and Method (continued) Results and DiscussionSISCAPA® (Stable Isotope Standards and Capture by
Anti-Peptide Antibodies) assays are a popular tool for
validation of candidate protein biomarkers. They are
used in research and in the clinic as a highly-specifi c
and high-throughput alternative to immunoassay
and traditional mass spectrometry. We report here
an implementation of 96-well protein digestion and
ConclusionReferences
As a sample enrichment technique, SISCAPA was successfully incorporated in a 11-plex LC-MRM assay to determine the abundance of 11 proteins in human serum. The 96-well format digestion on a PCR thermal cycler and automated liquid handling workstation allows for high-throughput quantitative analyses of multiple target proteins in biological samples by LC-MRM. In a multiplexing SISCAPA-LC-MRM assay, optimization of the coupling of magnetic beads with monoclonal antibodies is critical for a successful outcome of sample enrichment and subsequent LC-MRM detection.
Figure 1 High-throughput multiplexed SISCAPA-LC-MRM workfl ow
Table 1 Peptide fragment transitions monitored in the 11-plex MRM
Table 2
Figure 2 Representative chromatograms for Peptide 3
Figure 3 Representative chromatograms for Peptide
SISCAPA enrichment produced clean background (see Figures 2 and 3 for representative chromatograms). As a result, the signal-to-noise ratio was much improved compared to extractions such as protein precipitation and solid-phase extraction (data not shown). Improved signal-to-noise ratios translate into lower levels of detection/quantitation thus yielding higher sensitivity of a LC-MRM assay. In the present study, lower limit of quantitation (LLOQ) was achieved below 1 ng/mL for the majority of the 11 analytes (data not shown).
In the absence of protein standards, we used blank matrix samples to calculate the variability of endogenous tryptic peptides across four runs. The coeffi cient of variation (CV) varied signifi cantly both within and between runs depending on the analytes (Table 2). In general, weaker signals displayed greater CVs within runs (e.g. Peptides 3, 8, 10, 11 and 13 as shown in Table 2). We observed greater inter-assay CVs for Peptides 4 (34%) and 12 (40%) even though the intra-assay CVs were 7 –
12% and 9 – 13%, respectively. The greater inter-assay CVs for Peptides 4 and 12 were probably a refl ection of the varied digestion effi ciency and/or extraction between runs, which highlights the importance of including protein QC samples in MRM assays, whenever possible, so the variation in digestion effi ciency/extraction can be addressed.
We compared the coupling of magnetic beads with the mAbs in a cocktail mixture and individual mAbs. Two of the 11 mAbs (SAT-71 and SAT-78) failed to produce signals following the cocktail coupling, while the individually coupled bead-mAb complexes produced signals for all the 11 peptides (data not shown). We suspect that SAT-71 and SAT-78 were out-competed by others in the cocktail coupling experiment. In the present study, we performed the individual bead-mAb coupling and combined the individually coupled bead-mAb complexes prior to use to ensure that all mAbs were coupled to beads and all target peptides were pulled down during sample enrichment.
SISCAPA mediated sample enrichment for 11-plex
targeted protein quantifi cation by LC-MS/MS.
The results are encouraging with respect to the
reduction of background complexity leading to
improved signal-to-noise ratio and sensitivity, albeit
rather variable in reproducibility across multiple
runs, depending on the analytes.
Under the direction of SISCAPA Assay Technologies, Inc. (SAT; Washington, DC), synthetic proteotypic peptide surrogates from each biomarker protein were synthesized, coupled to keyhole limpet hemocyanin as carrier, and the conjugates were used to immunize rabbits for derivation of rabbit monoclonal antibodies (RabMAbs) by Epitomics, Inc. (Burlingame, CA). To select hybridomas secreting anti-peptide antibodies, hybridoma supernatants from each fusion were tested by SAT in peptide ELISA (Razavi et al., 2011). Positive rabbit hybridoma supernatants were subsequently screened for high affi nity antibodies using a combination of immunoscreening-mass spectrometry (Razavi et al., 2011) and surface plasmon resonance (Pope et al., 2009) assays under conditions that measure monovalent
interactions (refl ecting antibody affi nity rather than avidity). Antibodies that were able to bind a target peptide from solution phase and retain it during washing for a minimum of 10 minutes were identifi ed, as required for use in high-sensitivity SISCAPA assays. The selected antibodies were produced in milligram quantities by recombinant expression at Epitomics and kinetic analysis of antibody-peptide binding was subsequently performed by surface plasmon resonance at SAT. All of the peptide-specifi c RabMAb reagents exhibited high affi nity with low dissociation constants. Finally, the recombinant RabMAbs were characterized in multiplex SISCAPA assays using human plasma digest matrix, and the amount of each antibody was optimized for use in peptide capture.
Anti-peptide monoclonal antibodies
Dynabeads and individual mAb coupling
I. Prepare Dynabeads® (Dynal, Inc.) a. Resuspend beads by vortexing vial > 30sec while tilting and rotating vial until no material is settled on the bottom of the container. b. Transfer 600µL (1.5mg) Dynabeads to a 2mL polypropylene (PPE) tube. c. Place the tube on the magnet to separate the beads from solution and remove the supernatant. d. Remove the tube from the magnet.
II. Wash Beads (2X) a. Add 1000µL of 1xPBS+0.03%CHAPS to the tube, cap and vortex gently. b. Place the tube on the magnet to separate the beads from solution and remove the supernatant. c. Repeat wash step (II.a and II.b). d. Transfer washed beads from the 2mL polypropylene (PPE) tube to a 15mL polypropylene (PPE) conical tube.
III. Bind mAb (individual mAb coupling)
a. Add 240µL mAb (in 1xPBS+0.03%CHAPS) to the 15mL tube containing the beads. b. Add an additional 1.92 mL 1xPBS+0.03%CHAPS to the 15mL tube containing beads to dilute to a fi nal volume of about 6mL including mAb volumes, cap and wrap capped tube with parafi lm to prevent leakage. c. Incubate at room temperature on rotational shaker for 1 hour.
IV. Bead-mAb complex Storage
a. Store bead-mAb complex in the 15mL tube at 4°C until needed.
Sample tryptic digestion (performed on GeneAmp® PCR System 9700 (Roche Molecular Systems, Inc.))
1. Sample volume: 135 µL (containing 4 µL of human serum, internal standard peptides, appropriate amount of calibrator peptides, in 67.5 mM ammonium bicarbonate) 2. Denaturation: 95oC, 10 min. 3. Quenching: 4oC, 10 min. 4. Reduction: 13 mM DTT, 50oC, 30 min. 5. Alkylation: 23 mM Iodoacetamide, 25oC, 30 min. 6. Digestion: 5 µg Trypsin (enzyme:substrate = 1:60, w/w), 37oC, 12-16 hours
Sample enrichment (performed on Hamilton Microlab® Star liquid handling workstation)
1. Combine the 11 individually coupled bead-mAb complexes to create bead-mAb cocktail. 2. Manually add 100µL bead-mAb cocktail to wells in 2mL 96-well NUNC plate using a multi channel pipette. 3. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the
supernatant. 4. Add entire digested sample (~135µL) from PCR plate to the 2mL 96-well NUNC plate containing washed
beads. 5. Add 500µL of 1XPBS+0.03%CHAPS to each well, cap plate and incubate for 1 hour at room temp using
the Vibrax VXR Basic at 900-1000rpm. 6. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove
the solvent. 7. Wash wells containing beads with 600µL of 1xPBS+0.03%CHAPS. 8. Shake using robotic shaker for 2 minutes at 1000 rpm. 9. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the
wash solvent. 10. Wash wells containing beads with 600µL of 1xPBS+0.03%CHAPS a second time. 11. Shake using Eva shaker for 2 minutes at 1000 rpm. 12. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution and remove the
wash solvent. 13. Add 125µL of 0.1% formic acid in water + 0.03% CHAPS to each well for peptide elution. 14. Cap plate and place on Eva shaker for 5 minutes at 1000 rpm. 15. Place the 2mL 96-well NUNC plate on the magnet to separate the beads from solution. 16. Remove the elution solvent and transfer into a 1mL 96-well NUNC plate and cap for analysis.
Liquid chromatography
System: Shimadzu CBM-20A, LC-20ADColumn: Agilent AdvanceBio® Peptide Map 2.1 x 100mm 2.7-micron 600BarColumn Temp: 40oCMPA: water with 0.1% formic acidMPB: acetonitrile with 0.1% formic acidWash 1: 60/30/10 acetonitrile/isopropanol/acetoneWash 2: 75/25 water/acetonitrile with 1% formic acidTotal Flow: 0.3 mL/minGradient: 0.8 min 0-20%B, 0.8 min 20-40%B, 0.4 min 40%B, 0.2 min 40-65%B, 0.6 min 65-95%B, 0.7 min 95%B, 0.5 min 95-0%B, 1 min 0%B (total 5 min.)
Mass spectrometry
Instrument: AB Sciex® API 5000Polarity: PositiveScan Type: MRMIon Source: Turbo SprayIS: 1500TEM: 500DP: 100CEM: 2500
Table 2 Intra- and Inter-assay variability across four runs. Variability was based on the peak area ratios of 11 endogenous tryptic peptides in blank matrix samples (13 blank matrix samples per run). The intra- and inter-assay CV (%) was calculated for each peptide.
Morteza Razavi, Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson. MALDI Immunoscreening (MiSCREEN): A Method for Selection of Anti-peptide Monoclonal Antibodies for Use in Immunoproteomics. Journal of Immunological Methods (2011); 364:50-64.
Matthew E. Pope, Martin V. Soste, Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson. Anti-peptide antibody screening: selection of high affi nity monoclonal reagents by a refi ned surface plasmon resonance technique. Journal of Immunological Methods (2009); 341:86-96.
