Thermo Scientific Mass Spectrometric Immunoassay (MSIA)

Transcription

1 INSTRUCTIONS FOR USE OF PRODUCTS Thermo Scientific Mass Spectrometric Immunoassay (MSIA) Demonstration Protocol for Affinity Purification and Analysis of Human Insulin from Human Plasma Utilizing MSIA Insulin D.A.R.T. S, MSIA TM Versette Automated Liquid Handler and Thermo Scientific Q Exactive LC/MS System Product Number Description MSIA Insulin D.A.R.T. S containing 1 rack of 96 tips MSIA Insulin D.A.R.T. S containing 1 pack of 24 tips. 650-MSIA MSIA Versette Automated Liquid Handler Table of Contents 1. Description 2 2. Important Product Information 2 3. MSIA Workflow for Qualitative and Quantitative Analysis of Insulin 3 A. Equipment and Materials Required 3 B. Versette Insulin MSIA Program 5 1. Verify Essential Settings 5 2. Verify the Analyte Capture Settings 7 3. Verify the Elution Settings 8 C. Preparing Reagents 9 D. Affinity Capture of Insulin Preparation of Reagent Plates Preparation of Insulin Standard Curve and Controls Perform the MSIA Affinity Capture of Insulin 12 E. Elution of Immuno-Captured Insulin 13 F. LC/MS Conditions for Analysis of Insulin 14 G. Mass Spectrometry Conditions Data Analyses Expected Results MSIA Workflow Ordering Information Description 2155 E. Conference Dr., STE msia.info@thermofisher.com Tempe, AZ USA Page 1

2 The assessment of native insulin and therapeutic analog levels is important for medical, sports doping, and forensic applications. In general, insulin analogues have modified primary sequences compared to native human insulin, resulting in reduced or no cross- reactivity with insulin assays used in clinical laboratories 1. To reduce the bias of insulin assays, a universal extraction method is required for simultaneous extraction of native and therapeutic insulin with subsequent targeted quantification by LC-MS for increased specificity. A mass spectrometric immunoassay (MSIA ), a multiplexed assay for intact insulin and its analogues that couples immuno-enrichment provided by insulin D.A.R.T. S with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented. Reference 1. Parfitt C, Church D, Armston A, Couchman L, Evans C, Wark G, McDonald TJ. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogues. In Press. 2. Important Product Information Storage Conditions: MSIA Insulin D.A.R.T. S may be stored at 4 C for up to 1 year from the date of purchase. This product should never be frozen. The MSIA Insulin D.A.R.T. S are intended for single use only. MSIA Insulin D.A.R.T. S are not intended for the transference or measurement of liquids. This product is intended for micro-scale analyte purification prior to mass spectrometric detection. Technical literature is available at: msia.info@thermofisher.com Contact Sales: Contact Support: Tempe, AZ USA Page 2

3 3. MSIA Workflow for Qualitative and Quantitative Analysis of Insulin This workflow has been optimized for the extraction and analysis of Insulin from human donor plasma. While this protocol may be utilized as a model for other MSIA protein extractions it is recommended that the conditions of this protocol are optimized individually for each specific analyte and antibody. 3.A. Equipment and Materials Required Equipment and Software MSIA Versette Automated Liquid Handler (Thermo Scientific, Product No. 650-MSIA) Set of Finnpipette F1 Adjustable-Volume Pipettes (Thermo Scientific, Product No ) or equivalent ProSwift RP-4H LC Columns 1 x 250 mm I.D. (Thermo Scientific, Product No ) Liquid Chromatography System capable of flow rates of ml/min Column heater compatible to 250 mm length LC column and capable of 60 C Auto Sampler System with 100µL Syringe and 100µL Sample Loop with the ability to accommodate 100 µl sample volumes and 96-well plates Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer Thermo Scientific XCalibur Software, Version 2.2 Thermo Scientific Pinpoint Software Additional Materials for the Affinity Capture of Insulin MSIA D.A.R.T. S, Insulin, 24-Pack (Thermo Scientific, Product No ) Human Donor Plasma (optional) Insulin, Human Recombinant (Gibco, Product No ) 4 [D10]Leu Insulin (Human) (0.02 mg vial), (Peptides International, Product No. PLP-3404-v) BupH Modified Dulbecco's PBS Packs (PBS); 10mM PBS ph 7.4 (Thermo Scientific, Product No ) Water, Optima LC/MS Grade (Fisher Chemical, Product No. W6) Tempe, AZ USA Page 3

4 Bovine Serum Albumin (BSA), Fraction V, Cold-ethanol Precipitated, (Fisher BioReagents, Product No. BP ) Pierce Octyl beta-glucoside (NOG), (Thermo Scientific, Product No. PI-28310) Acetic Acid, Glacial (Certified ACS Plus), (Fisher BioReagents, Product BP ) Nunc 2.0mL DeepWell Plates with Shared-Wall Technology (Thermo Scientific, ) Nunc 96-Well Polypropylene Plates, 500µL (Thermo Scientific, ) Additional Materials for Insulin Elution Trifluoroacetic Acid, Optima LC/MS Grade (Fisher Chemical, Product No. A116) Acetonitrile, Optima LC/MS Grade (Fisher Chemical, Product No. A996) ACTH [1-24] Peptide, (ABBIOTEC, Product No ) Materials for Insulin LC/MS Analysis Formic Acid, Optima LC/MS Grade (Fisher Chemical, Product No. A117) Water, Optima LC/MS Grade (Fisher Chemical, Product No. W6) Acetonitrile, Optima LC/MS Grade (Fisher Chemical, Product No. A996) Tempe, AZ USA Page 4

5 3.B. Versette Insulin MSIA Programs The Insulin MSIA Versette Program may be downloaded at: 3.B.1. Verify Essential Settings for the Insulin MSIA Versette Program The Verification Steps are for initial set up of a newly downloaded MSIA Versette program should not need to be repeated once the system is running the program properly. 1. Open the Insulin MSIA Versette program with the ControlMate Software and uncollapse the Main Sequence. 2. Use Graphic 1 to verify that the Main Sequence settings and the Pipette Head & Tips Settings (Upper Right of Graphic 1) are properly set for the Insulin MSIA Versette program. Refer to Steps 3 & 4 below to adjust the Pipette Head & Tips Settings otherwise Steps 3 & 4 may be skipped. Graphic 1: Insulin MSIA Versette Program Main Sequence Settings and the Pipette Head & Tips Settings Tempe, AZ USA Page 5

6 3. To change the Pipette Head and Tips settings select the Change Pipette Head and Tips option from the Add-Ins drop down menu located at the Top-Mid-Left of the ControlMate window. 4. Adjust the Change To settings in the Addin: Change Pipette Head and Tips window to reflect the settings of Graphic 2 below. Then hit the Start button to initiate the changes. Graphic 2: Pipette Head & Tips Settings for the Insulin MSIA Versette Protocol. Tempe, AZ USA Page 6

7 3.B.2. Verify the Analyte Capture Settings 1. Uncollapse the group named Analyte Capture and the subsequent groups that make up the Analyte Capture Steps. Individually compare the program values with Graphic 4. Specifically double check that the correct Vessel Type is selected under each Move to Stage function. i. All rinses should utilize the 96 MicroWell Plates U Bottom PP vessel and the Insulin Samples should utilize the 96 DeepWell Plates 2.0ml U Bottom PP vessel as displayed in Graphic 4. ii. Note: Graphic 4 shows two rinses in their collapsed state. These two rinses share identical settings to the rinse performed directly before them with the exception of the Stage. Tempe, AZ USA Page 7

8 Graphic 4: Analyte Capture Settings for the Insulin MSIA Versette Protocol. 3.B.3. Verify the Elution Settings 1. Uncollapse the group named Elution and the subsequent groups that make up the Elution Steps. Individually compare the program values with Graphic 5. Specifically double check that the correct Vessel Type is selected under each Move to Stage function. Tempe, AZ USA Page 8

9 Graphic 5: Elution Settings for the Insulin MSIA Versette Protocol. 3.C. Preparing Reagents Prepare 10mL of 150mM NOG in 10mM PBS Dissolves 0.439g NOG in 7.5mL of 10mM PBS. Adjust final volume to 10mL with 10mM PBS. Prepare 10mL of 50g/L BSA in 10mM PBS Dissolves 0.5g BSA in 7.5mL of 10mM PBS. Adjust final volume to 10mL with 10mM PBS. Prepare 100µL of 10% Trifluoroacetic Acid (v/v) Add 10µL of Trifluoroacetic Acid to 90µL of Water, Optima. Prepare 250µL of 1% Acetic Acid (v/v) Add 2.5µL of Acetic Acid to 247.5µL of Water, Optima. Tempe, AZ USA Page 9

10 3.D. Affinity Capture of Insulin The procedure below outlines the preparation of a 5-point curve and three controls for the quantitative analysis of Human Insulin making use of 8 positions of the Versette s 96-channel head and utilizing eight Insulin D.A.R.T. S. If initial testing of the system is necessary prior to running a curve then prepare only 1-3 D.A.R.T. S and reduce the reagents appropriately for each step. The recommended sample concentration for testing the MSIA Workflow is 960pM of Insulin standard in 500µL of 50g/L BSA in 10mM PBS. Further sample preparation is described in detail below within the protocol. 3.D.1. Preparation of Reagent Plates for the Analyte (Insulin) Capture Use the color coded plate maps represented below to add the analyte capture reagents to the corresponding wells of six 96-well microplates. Reference Table 1 for the color key: Table 1: Key Code for the 96-Well Plate Maps for the MSIA Insulin Versette Workflow Representative Reagent Reagent Texture Plate Versette Stage Rinse Buffer Squares 200µL of PBS 1, 3, 4 1, 3, 4 Water Dots 200µL of Optima LC/MS Grade Water 5, 6 5, 6 Sample* Solid Gray *Sample preparation specified in Step 3.D.II. 2 2 Tempe, AZ USA Page 10

11 3.D.2. Preparation of Insulin Standard Curve and Controls 1. Prepare the 4 [D10]Leu Insulin standard at a concentration of 20µM by reconstituting the 24µg vial with 205µL of 1% Acetic Acid. The 4 [D10]Leu Insulin Standard is intended to act as an internal reference standard normalizing the samples as they undergo the affinity capture steps. 2. Prepare the sample dilutions for the 4 [D10]Leu Insulin and the rhuman Insulin Standard Curve and Controls by following the dilution table outlined in Table 2.1 and Table Note: Following preparation of the samples, aliquot the remaining 4 [D10]Leu Insulin and the rhuman Insulin standards and store at -20 C or below. Table 2.1 Quick Reference Dilutions for 4 [D10]Leu Insulin IRS 4 [D10]Leu Insulin Internal Reference Standard Dilutions Final Volume and Concentration of 4 [D10] Leu Insulin Combine Initial 4 [D10]Leu Insulin Concentration 150mM NOG in 10mM PBS (µl) 200µL of 1µM 10µL of 20µM µL of 100nM 10µL of 1µM µL of 10nM 20µL of 100nM µL of 1nM 100µL of 10nM mL of 100pM 440µL of 1nM Tempe, AZ USA Page 11

12 Table Quick Reference Dilutions for rhuman Insulin Standard Curve and Controls Final Volume and Concentration of rhuman Insulin Combine rhuman Insulin Standard Curve Initial rhuman Insulin Concentration 50g/L BSA in 10mM PBS (µl) 71.7µL of 96µM 10µL of 688.7µM (4g/L) µL of 9.6µM 10µL of 96µM µL of 960nM 10µL of 9.6µM µL of 96nM 10µL of 960nM µL of 9.6nM 20µL of 96nM µL of 960pM 100µL of 9.6nM µL of 240pM 325µL of 960pM µL of 60pM 250µL of 240pM µL of 15pM 250µL of 60pM µL of 7.5pM 300µL of 15pM Final Volume and Concentration of rhuman Insulin rhuman Insulin Standard Controls Combine Initial rhuman Insulin Concentration 50g/L BSA in 10mM PBS (µl) 2040µL of 50pM* 425µL of 240pM *The rhuman Insulin Standard Controls may be substituted for Normal Human Plasma. If utilizing Human Plasma as the controls it is not necessary to spike in any rhuman Insulin standard. 3. Add 500µL of 100pM 4 [D10]Leu Insulin Standard in 150mM NOG in 10mM PBS to each of the 8 wells in column 2 of the Sample Plate. 4. Follow Table 3 to add 500µL of each of the Insulin standard curve and control samples to their corresponding wells in column 2 of the Sample Plate. Table 3 Plate Map for Insulin Standard Curve and Control Samples Insulin Standard Concentration 7.5pM 15pM 60pM 240pM 960pM Control 1 50pM Control 2 50pM Control 3 50pM Well H1 H2 H3 H4 H5 H6 H7 H8 Tempe, AZ USA Page 12

13 3.D.3. Preparation of the Versette for the Affinity Capture of Insulin 1. Refer to Graphic 6 for the 6 positions that make up the Versette stage. Graphic 6 Numbering of Versette Stages 2. Secure plates 1-6 onto their corresponding positions on the Versette stage as specified in stable 1 corresponding and Plate Map (Section 3.D.1). Plates should be positioned with well A1 located at the upper left of the Stage. 3. Load eight MSIA Streptavidin D.A.R.T. S into the 96-D.A.R.T. S Magazine using positions H1-H8 as depicted in Graphic 7. Then fill the remaining empty magazine positions with blank or cut D.A.R.T. S in order to balance the pressure the Versette will be applying to them. Graphic 7 Preparation of MSIA D.A.R.T. S Magazine Tempe, AZ USA Page 13

14 4. Load and run the Insulin_Capture & Elution Versette program. When prompted, insert the loaded D.A.R.T. S Magazine into the 96-Channel Versette Head as depicted in Graphic 8. The magazine will click into place when inserted correctly. Click the continue button and the program should take ~1 hour and 15 minutes to complete. Following the affinity capture steps the Versette program will pause allowing for the stages to be set up for the Elution step. Graphic 8 Inserting D.A.R.T. S Magazine into Versette 96-Channel Head 3.E. Elution of Immuno-Captured Insulin 1. Prepare the Elution Solvent fresh by combining 24µL of 10% LC-MS Grade Trifluoroacetic Acid + 198µL of LC-MS Grade Acetonitrile + 4.5µL of 2mg/mL ACTH µL of LC-MS Grade Water. For a final volume of 600µL of 15µg/mL ACTH Standard in 0.4% Trifluoroacetic Acid in 33% Acetonitrile. 2. The elution will be performed in a 96-Well Polypropylene Plate, 500µL (Elution Plate). Add 63µL of the Elution Solvent to wells H1-H8 of the Elution Plate as depicted in Graphic 9. Tempe, AZ USA Page 14

15 3. Remove the Affinity Capture Plates from the Versette Stages. Place the Elution Plate onto the Versette Stage Click the Ok button to continue the Elution steps of the program, which should take ~10 minutes. 5. Following completion of the Versette program, remove the elution plate and add 21µL of Water, Optima to each of the 8 eluate(s). This will reduce the Acetonitrile concentration to ~25% improving conditions for loading onto LC. 6. Seal the plate. Mix the samples briefly (~30 seconds). Then spin the samples down. The eluates are ready for LC-MS analysis of Intact Insulin and should be directly loaded onto the LC-MS from the elution plate. 3.F. LC/MS Conditions for Analysis of Insulin LC Conditions: Column Temperature: 60 C Mobile Phase A: 0.2% Formic Acid in Water Mobile Phase B: 0.2% Formic Acid in Acetonitrile Gradient Table: Time (mins) % Mobile Phase B Flow Rate (µl/min) Tempe, AZ USA Page 15

16 3.G. Mass Spectrometry Conditions The optimization of the Analysis of Insulin was performed on a Thermo Scientific Q Exactive Mass Spectrometer with a Thermo Scientific Dionex UltiMate 3000 UHPLC System. Equivalent instruments to the Q Exactive may require further optimization of the MS Conditions. All data should be acquired using a Thermo Scientific Q Exactive Orbitrap mass spectrometer operated in full scan MS mode. Full scan MS data was acquired with a resolution setting of 70,000 (at m/z 200) and using a mass range of Da. 4. Data Analyses The benefits of acquiring HR/AM MS data is the ability to utilize the most abundant precursor charge states per insulin analog and the most abundant isotopes per each charge state to provide both qualitative validation, as well as quantitative measurement for each insulin analog (Proteomics 2014, 14, ). The summation of the five most abundant isotopes per each of the +5 and +6 precursor charge states were combined to provide the total AUC (Area Under the Curve) for the rhum Insulin. Only the summation of the five most abundant isotopes for the +5 precursor charge state is necessary for the 4 [D10]Leu Insulin, the internal reference standard. Reference Table 4 for a listing of the precursor m/z values utilized. Table 4 A list of the charge states and precursor ions summed from the MS data to provide the AUC value for the recombinant human insulin and the 4 [D10]Leu heavy insulin. rhum Insulin + 5 Charge State + 6 Charge State Precursor m/z values [D10]Leu Insulin Precursor m/z values Charge State Charge State Tempe, AZ USA Page 16

17 5. Expected Results The rhum Insulin AUC values are then divided by the 4 [D10] Insulin AUC values, normalizing the data and providing the file area ratio. The file area ratio for each concentration is plotted and the equation of the resulting line is used to determine the experimental concentration of each unknown. Example data for the 5 point standard curve and the three controls are provided below (Tables 5 & 6 and Graphic 3). Table 5 Concentration (pm) File Area Ratio (AUC normalized by IRS) Experimental Concentration (pm) E E E E E Table 6 Control File Area Ratio (AUC normalized by IRS) Experimental Conc (pm) CVs Accuracy E % 0.62% E E Tables 5 & 6 Data resulting from performing the Insulin MSIA workflow utilizing Insulin D.A.R.T. S and Finnpipette Novus i for the immunopurification of rhuman Insulin from BSA. Analysis was performed on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with a Dionex UltiMate 3000 UHPLC System. The File Area Ratio was achieved by normalizing the rhuman Insulin AUC (Area Under the Curve) with the [D10]Leu Insulin AUC from the mass spectra. The resulting File Area Ratios were then utilized to produce the equation of the line to calculate the Experimental Concentration 3.0E+01 rhuman Insulin Curve File Area Ratio (Normalized by IRS) 2.5E E E E E E+00 y = x R² = rhuman Insulin Concentration (pm) Graphic 3 Example rhuman Insulin Standard Curve. Data taken from Anti-Insulin MSIA DARTS Reproducibility Study: Inter-Day 1; Curve 1 Tempe, AZ USA Page 17

18 6. Ordering Information MSIA D.A.R.T. S for Immunoaffinity Capture Compatible with the Thermo Scientific Versette Automated Liquid Handler and Thermo Scientific Finnpipette Novus i Multichannel Electronic Pipette Cat. No. Description Packaging 991CUS02 300µl MSIA D.A.R.T. S, Custom Pack of 96 units 991PRT11 300µl MSIA D.A.R.T. S, Protein A Pack of 96 units 991PRT12 300µl MSIA D.A.R.T. S, Protein A Pack of 24 units 991PRT13 300µl MSIA D.A.R.T. S, Protein G Pack of 96 units 991PRT14 300µl MSIA D.A.R.T. S, Protein G Pack of 24 units 991PRT15 300µl MSIA D.A.R.T. S, Protein A/G Pack of 96 units 991PRT16 300µl MSIA D.A.R.T. S, Protein A/G Pack of 24 units 991STR11 300µl MSIA D.A.R.T. S, Streptavidin Pack of 96 units 991STR12 300µl MSIA D.A.R.T. S, Streptavidin Pack of 24 units µl MSIA D.A.R.T. S, Insulin Pack of 96 units µl MSIA D.A.R.T. S, Insulin Pack of 24 units 991R 300 µl MSIA D.A.R.T. S, Reloadable Rack 1 reloadable rack, D.A.R.T. S are not included Automated Liquid Handling Platform Cat. No. Description 650-MSIA MSIA Versette Automated Liquid Handler Multichannel Pipettes and Pipette Stand Cat. No. Description Packaging 991S Finnpipette Novus i Adjustable Pipette Stand 1 pipette stand 991SP12 Finnpipette Novus i Electronic 12-Channel Pipette, µl and Pipette Stand 1 pipette and 1 pipette stand Liquid Chromatography Cat. No. Description Thermo Scientific Dionex UltiMate 3000 UHPLC System ProSwift RP-4H Monolith Column, 1.0 x 250 mm Mass Spectrometry and Software Description Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer Thermo Scientific Pinpoint Software Thermo Scientific XCalibur Software Thermo Scientific Protein Deconvolution Software, Version 3.0 with the ReSpect algorithm thermoscientific.com/msia Products are intended for research use only Thermo Fisher Scientific Inc. All rights reserved. North America: info.sandiego@thermofisher.com Outside North America: info.sandiego@thermofisher.com Tempe, AZ USA Page 18