demonstration protocol for affinity purification and lc/ms ......derivatization of protein a/g...

INSTRUCTIONS FOR USE OF PRODUCTS

Thermo Scientific™ Universal Mass Spectrometric Immunoassay (MSIA)

Demonstration Protocol for Affinity Purification and LC/MS Analysis of Digested IGF-I from Human Plasma Utilizing MSIA Universal Protein A/G D.A.R.T.s, Versette™ Automated Liquid Handler, and Thermo Scientific™ TSQ Vantage™ LC/MS System

991PRT15 991PRT16

Product Number Description

991PRT15 MSIA D.A.R.T.’S Protein A/G containing 1 rack of 96 tips.

991PRT16 MSIA D.A.R.T.’S Protein A/G containing 1 pack of 24 tips.

650-MSIA MSIA™ Versette™ Automated Liquid Handler Table of Contents

1. Important Product Information 2 2. Universal MSIA Workflow for Quantitative Analysis of Digested IGF-I 2

A. Equipment and Materials Required 2 B. Versette Protein A, G, A/G MSIA Program 4

1. Verify Essential Settings 4 2. Verify the Affinity Ligand Capture Settings 7 3. Verify the Analyte Capture Settings 8 4. Verify the Elution Settings 9

C. Preparing Reagents 11 D. Derivatization of Protein A/G D.A.R.T.’S with IGF-I Antibody 12

1. Preparation of Reagent Plates 12 2. Preparation of the Versette 13

E. Analyte (IGF-I) Capture 15 1. Preparation of Reagent Plates 15 2. Preparation of IGF-I Standard Curve and Controls 16 3. Perform the Protein A/G MSIA Analyte (IGF-I) Capture 18

F. Elution of Immuno-Captured IGF-I 18 3. Ordering Information 20

Thermo Scientific North America Toll Free: +1 800 995 2787 www.thermoscientific.com/msia MSIA – Mass Spectrometric Immunoassay Outside North America Toll Free: +1 858 453 7551 2155 E. Conference Dr., STE 104 E-Mail: [email protected] Tempe, AZ 85284-2604 USA Page | 1

mailto:[email protected]

1. Important Product Information

• Storage Conditions: MSIA Protein A/G D.A.R.T.’S should be stored at 4 °C for up to 1 year

from the date of purchase. This product should never be frozen.

• The MSIA Protein A/G D.A.R.T.’S are intended for single use only.

• MSIA Protein A/G 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.

2. Universal MSIA Workflow for Quantitative Analysis of Digested IGF-I

This workflow is a generalized protocol that utilizes IGF-I from human plasma as an example. While

this protocol may be utilized as a model for other Universal MSIA protein assays it is recommended that

the conditions of this protocol be individually optimized for each specific analyte and antibody.

2.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.

4700850) or equivalent

• Accucore™ aQ HPLC Columns, 2.6µm particle size; 100 x 2.1mm I.D. (Thermo Scientific™,

Product No. 17-326-102130)

• Thermo Scientific™ TSQ Vantage™ Triple Quadrupole Mass Spectrometer

• Liquid Chromatography System capable of flow rates of 0.24-0.8 mL/min

• Liquid Chromatography System with a column heater capable of 35 °C

• Auto Sampler System with a 250µL Syringe and 250µL Sample Loop with the ability to

accommodate 96-well plates

Technical literature is available at: www.thermoscientific.com/msia Email: [email protected]

Contact Sales: +1 800 995 2787 Contact Support: +1 800 345 0206



http://www.thermoscientific.com/msia

• Express SC250EXP SpeedVac™ Concentrator System (Thermo Scientific™, Product No.

SC250EXP-115)

• Thermo Scientific™ XCalibur™ Software

• Thermo Scientific™ Pinpoint Software

Materials for the Derivatization of Protein A/G D.A.R.T.’S with IGF-I Antibody

• MSIA D.A.R.T.’S, Protein A/G, 24-Pack (Thermo Scientific™, Product No. 991PRT16)

• Anti-Human IGF-I (PeproTech, Product No. 500-P11)

• MES (Fine White Crystals), (Fisher BioReagents, Product No. BP300-100)

• BupH™ Modified Dulbecco's PBS Packs (PBS); 10mM PBS pH 7.4 (Thermo Scientific™, Product

No. 28374)

• Nunc™ 96-Well Polypropylene Plates, 500µL (Thermo Scientific™, 267245)

Additional Materials for Affinity Purification of IGF-I

• Human Donor Plasma (optional)

• Recombinant Human Insulin-like Growth Factor I (IGF-I), (Cell Sciences®, Product No.

CRI500A)

• IGF1 N15 Labeled Human Recombinant, (ProSpec, Product No. CYT-128)

• Water, Optima™ LC/MS Grade (Fisher Chemical, Product No. W6)

• Bovine Serum Albumin (BSA), Fraction V, Cold-ethanol Precipitated, (Fisher BioReagents™,

Product No. BP1605100)

• Sodium Dodecyl Sulfate (SDS) (White Powder/Electrophoresis), (Fisher BioReagents™, Product

No. BP166-100)

Additional Materials for IGF-I Elution

• Trifluoroacetic Acid, Optima™ LC/MS Grade (Fisher Chemical, Product No. A116)

• Acetonitrile, Optima™ LC/MS Grade (Fisher Chemical, Product No. A996)

• 96-well Robotic PCR Plate (Thermo Scientific™, AB-1300)



2.B. Versette Protein A, G, A/G Programs

2.B.1. Verify Essential Settings for the Protein A, G, A/G Versette Program

1. Open the Protein A, G, A-G MSIA_Complete Workflow_GV 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 Protein A, G, A-G

MSIA_Complete Workflow_GV Versette program. Refer to Steps 3 & 4 below to

adjust the Pipette Head & Tips Settings otherwise Steps 3 & 4 may be skipped.

The Verification Steps are for initial set up of a newly downloaded MSIA Versette program and should not need to be repeated once the system is running the program properly.

The Protein A, G, A-G MSIA_Complete Workflow Versette Program may be downloaded at: www.thermoscientific.com/msia

For the pre-analytical Trypsin digestion and LC/MS/MS protocol for IGF-I please refer to the following application note:

A Universal Mass Spectrometric Immunoassay (MSIA™) Model System Based on Human Insulin-Like Growth Factor 1 (IGF1)

Technical literature is available at: www.thermoscientific.com/msia

Contact Support: +1 800 345 0206 Outside North America Toll Free: + 1 858 453 7551

Email: [email protected]





Graphic 1: Protein A, G, A/G MSIA Versette Program – Main Sequence Settings and the Pipette Head & Tips Settings

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 select the “Start” button to

initiate the changes.



Graphic 2: Pipette Head & Tips Settings for the Protein A, G, A/G MSIA Versette Program



2.B.2. Verify the Affinity Ligand Capture Settings

1. Uncollapse the group named “Affinity Ligand Capture” and the subsequent groups

that make up the Affinity Ligand 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 stages should utilize the “96 MicroWell Plates U Bottom PP” as displayed in

Graphic 4.

Graphic 4: Affinity Ligand Capture Settings for the Protein A, G, A/G MSIA Versette Program



2.B.3. 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 5. Specifically double check that the correct “Vessel Type” is

selected under each “Move to Stage” function.

i. All stages should utilize the “96 MicroWell Plates U Bottom PP” as displayed in

Graphic 5.

ii. Note: Graphic 5 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.



Graphic 5: Analyte Capture Settings for the Protein A, G, A/G MSIA Versette Program

2.B.4. Verify the Elution Settings a. Uncollapse the group named “Elution” and the subsequent groups that make up the

Elution Steps. Individually compare the program values with Graphic 6. Specifically

double check that the correct “Vessel Type” is selected under each “Move to Stage”

function.



Graphic 6: Elution Settings for the Protein A, G, A/G MSIA Versette Program



2.C. Prepare Reagents

• Prepare 50mL of 10mM MES, pH 5.0 → Dissolve 0.1g of MES in 47mL of LC/MS

Grade Water. Adjust the pH to 5.0. Bring the final volume to 50mL with LC/MS

Grade Water.

• Prepare 10mL of 10g/L BSA in 10mM PBS → Dissolve 0.1g of BSA in 8mL of

10mM PBS. Adjust final volume to 10mL with 10mM PBS.

• Prepare 33.3mL of 0.3% SDS in 10mM PBS (w/v) → Dissolve 0.1g of SDS in

30mL of 10mM PBS. Adjust final volume to 33.3mL with 10mM PBS.

• Prepare 100µL of 10% Trifluoroacetic Acid (v/v) → Add 10µL of Trifluoroacetic

Acid (v/v) to 90µL of LC/MS Grade Water. 100uL of 10% Trifluoroacetic Acid is

enough for 10 samples.



2.D. Derivatization of Protein A/G D.A.R.T.’S with IGF-I Antibody (Human)

2.D.1. Preparation of Reagent Plates

Use the color coded plate maps represented below to add the affinity ligand 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 Protein A/G MSIA Affinity Ligand (Human Anti-IGF-I) Capture

Reagent Representative

Texture Reagent Plate

Versette Stage

Rinse Buffer Lines 200µL of MES 1 1

Affinity Ligand Solution

Solid Black 100µL of 30µg/mL IGF-I Antibody (Human) in 10mM MES

2 2

Rinse Buffer Squares 200µL of PBS 3 3

The procedure below outlines the preparation of a 5-point curve and three controls for the quantitative analysis of IGF-I making use of 8 Protein A/G 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 1500 ng/mL of IGF-I standard in 100 µL of 10 g/L BSA in 10 mM PBS. Further sample preparation is described in

detail below within the protocol.



2.D.2. Preparation of the Versette for the Derivatization of Protein A/G D.A.R.T.’S with

IGF-I Antibody (Human).

1. Refer to Graphic 7 for the 6 positions that make up the Versette stage.

Graphic 7 – Numbering of Versette Stages

2. Secure plates 1-3 onto their corresponding positions on the Versette stage as specified

in Table 1 and Plate Map (Section 2.D.1). Plates should be positioned with well A1

located at the upper left of the Stage.

3. Load eight Protein A/G MSIA D.A.R.T.’S into the 96-D.A.R.T.’S Magazine using positions

H1-H8 as depicted in Graphic 8. 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 8 – Preparation of MSIA D.A.R.T.’S Magazine

4. Load and run the Protein A, G, A-G_MSIA_Complete Versette program. When prompted,

insert the loaded D.A.R.T.’S Magazine into the 96-Channel Versette Head as depicted in

Graphic 9. The magazine will click into place when inserted correctly. Click the

“Continue” button and the program should take ~25 minutes to complete the affinity

ligand capture. Then the program will pause allowing for stages to be setup for the

analyte capture steps.

Graphic 9 – Inserting D.A.R.T.’S Magazine into Versette 96-Channel Head



2.E. Analyte (IGF-I) Capture

2.E.1. Preparation of Reagent Plates 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 2 for the color key:

Table 2: Key Code for the 96-Well Plate Maps for the Protein A/G MSIA Analyte (IGF-I) Capture

Reagent Representative Texture

Reagent Plate Versette Stage

Rinse Buffer Squares 200µL of PBS 4, 6, 7 1, 3, 4

Sample* Solid Gray *Sample preparation specified in Step 2.E.2. 5 2

Water Dots 200µL of Optima™ LC/MS Grade Water 8, 9 5, 6



2.E.2. Preparation of IGF-I Standard Curve and Controls

1. Add 100µL of 0.3% SDS in 10mM PBS to wells H1-H8 (Reference Table 2 and

corresponding Plate Maps in Section 2.E.1) of the Sample Plate.

2. Prepare the N15 Labeled IGF-I standard at a concentration of 0.1mg/mL by

reconstituting the 10µg vial with 100µL LC/MS Grade Water.

3. Prepare the rHuman IGF-I at a concentration of 0.1mg/mL by reconstituting the

20µg vial with 200µL LC/MS Grade Water.

4. Prepare the sample dilutions for the N15 Labeled IGF-I as well as the rHuman IGF-I

Standard Curve and Controls by following the dilution table outlined in Table 3.

- Note: Following preparation of the samples, aliquot the remaining N15

Labeled IGF-I and the rHuman IGF-I and store at -20 °C or below.

The N15 Labeled IGF-I Standard is intended to act as an internal reference standard normalizing the samples as they undergo the affinity purification, reduction, alkylation, and digestion steps.



5. Add 20µL of 500ng/mL N15 Labeled IGF-I Standard in 10mM PBS to each of the 8 wells in column 5 of the Sample Plate.

6. Follow Table 4 to add 40µL of each of the IGF-I standard curve and control samples to their corresponding wells in column 5 of the Sample Plate.

Table 4 –

Plate Map for IGF-I Standard Curve and Control Samples

IGF-I Standard Concentration

Well

5ng/mL H1 25ng/mL H2

100ng/mL H3 500ng/mL H4

1500ng/mL H5 Controls 1-3; 50ng/mL H6-8

7. Allow the samples to incubate for 20 minutes at room temperature (20-25 °C).

Table 3 – Quick Reference Dilutions for N15 Labeled IGF-I IRS and rHuman IGF-I Standard Curve and Controls

N15 Labeled IGF-I Internal Reference Standard Dilutions

Final Volume and Concentration Combine

Initial N15 IGF-I Concentration 10mM PBS (µL)

200µL of 5µg/mL → 10µL of 0.1mg/mL + 190

200µL of 500ng/mL → 20µL of 5µg/mL + 180

rHuman IGF-I Standard Curve Final Volume and

Concentration Combine Initial rHuman IGF-I

Concentration 10g/L BSA in 10mM PBS (µL)

60µL of 15µg/mL → 9µL of 0.1mg/mL + 51

100µL of 1500ng/mL → 10µL of 15µg/mL + 90

300µL of 500ng/mL → 100µL of 1500ng/mL + 200




rHuman IGF-I Standard Controls Final Volume and

Concentration Combine Initial rHuman IGF-I

Concentration 10g/L BSA in 10mM PBS (µL)

150µL of 50ng/mL* → 75µL of 100ng/mL + 75 *The rHuman IGF-I 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 IGF-I standard.



2.E.3. Perform the Protein A/G MSIA Analyte Capture of IGF-I

1. Secure plates 4-9 onto their corresponding positions on the Versette stage as

specified in Table 2 and corresponding Plate Maps (Section 2.E.1). Plates should be

positioned with well A1 located at the upper left of the Stage.

2. Once the plates are properly staged, press the “Ok” button on the pause prompt.

This will continue the program performing the analyte capture step of the Protein

A/G MISA workflow. The program should take ~35 minutes to complete the analyte

capture and will pause allowing for stages to be setup for the elution step.

2.F. Elution of Immuno-Captured IGF-I

1. Prepare 450µL of the Elution Solvent (0.4% Trifluoroacetic Acid in 33% Acetonitrile)

fresh by combining the following:

• 18µL of 10% LC-MS Grade Trifluoroacetic Acid

• 150µL of LC-MS Grade Acetonitrile

• 282µL of LC/MS Grade Water.

2. The elution will be performed in a 96-Well Polypropylene Plate, 500µL (Elution Plate).

Add 50 µL of the Elution Solvent to wells H1- H8 of the Elution Plate as depicted in

the plate map below:



3. Remove the Analyte Capture Plates from the Versette Stages. Place the Elution Plate onto the Versette Stage 2.

4. Click the “Ok” button on the pause prompt to continue the Elution steps of the Protein A, G, A-G MSIA_Complete Workflow_GV Versette program, which should take ~1 minute.

5. The eluates containing intact IGF-I are ready for pre-analytical digesion.

For the pre-analytical Trypsin digestion and LC/MS/MS protocol for IGF-I please refer to the following application note:

A Universal Mass Spectrometric Immunoassay (MSIA™) Model System Based on Human Insulin-Like Growth Factor 1 (IGF1)

Technical literature is available at: www.thermoscientific.com/msia

Contact Support: +1 800 345 0206 Outside North America Toll Free: + 1 858 453 7551

Email: [email protected]




3. 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 991001096 300µl MSIA D.A.R.T.’S, Insulin Pack of 96 units 991001024 300µl 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, 30-300µl and Pipette Stand 1 pipette and 1 pipette stand

Liquid Chromatography Cat. No. Description Thermo Scientific™ Dionex™ UltiMate® 3000 UHPLC System 066640 ProSwift™ RP-4H Monolith Column, 1.0 x 250 mm Mass Spectrometry and Software Description Thermo Scientific™ TSQ Vantage Triple Stage Quadrupole Mass Spectrometer Thermo Scientific™ Pinpoint Software Thermo Scientific™ XCalibur™ Software

thermoscientific.com/msia Products are intended for research use only. © 2015 Thermo Fisher Scientific Inc. All rights reserved.

North America: +1 800 995 2787 ● [email protected] Outside North America: +1 858 453 7551 ● [email protected]



demonstration protocol for affinity purification and lc/ms ......derivatization of protein a/g...

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