INSTRUCTIONS

High-SelectTM SMOAC Protocol

Sequential enrichment of Metal Oxide Affinity Chromatography

A32993 A32992 2162623.0

Number Description
A32993 High-Select TiO2 Phosphopeptide Enrichment Kit
Kit Contents:
TiO2 Spin Tips, 24 each
Centrifuge Column Adaptors, 24 each
Wash Buffer, 1.8mL
Binding/Equilibration Buffer, 7.0mL
Phosphopeptide Elution Buffer: 7.0mL

A32992 High-Select Fe-NTA Phosphopeptide Enrichment Kit, 30 columns

Kit Contents:
Spin Columns, 30 columns, 200 μl of resin slurry per column
White Luer Plug (End Cap), 30 Plugs
Binding/Wash Buffer, 2 x 20 mL
Phosphopeptide Elution Buffer, 7.0 mL

Storage: Upon receipt store at 4°C. Product shipped with ice packs.

Workflow of the SMOAC method

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce

3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

Introduction
The Thermo Scientific™ Pierce™ TiO2 Phosphopeptide Enrichment Kit enables efficient isolation of phosphorylated
peptides from complex and fractionated protein digests for analysis by mass spectrometry (MS). Spherical porous
titanium dioxide (TiO2) resin spin tips combined with optimized buffers provide enhanced identification and enrichment
of phosphopeptides with greater than 85% specificity. The kit’s newly optimized protocol and buffers result in a higher
yield of phosphopeptides ready for direct MS analysis without the need for additional graphite or C18 clean up.
Mass spectrometry is a key tool for identifying sites of protein phosphorylation and quantifying phosphorylation changes.
However, MS analysis of protein phosphorylation is challenging due to the low stoichiometry, high hydrophilicity, poor
ionization and incomplete fragmentation of phosphopeptides. Because of the low relative abundance of phosphorylation
modifications in complex protein samples, enrichment is essential for successful MS analysis of phosphopeptides. The
improved Pierce TiO2 Phosphopeptide Enrichment Kit is compatible with our lysis, reduction, alkylation, digestion and
high pH reversed-phase peptide fractionation columns to provide a complete workflow for phosphopeptide enrichment.

Important Product Information

 The Thermo Scientific Pierce Mass Spec Sample Prep Kit for Culture Cells can be used to prepare peptide digest
samples. Urea-based lysis methods can also be used for the preparation of peptide sample. Please refer to protein
biology application note. [1]
 It is recommended to enrich phosphopeptides from lyophilized peptide samples free of detergents and salts. It is
imperatively important that desalted peptide samples are entirely dissolved in Binding Buffer for optimal results.
 Each spin tip can enrich phosphopeptides from 0.5mg to 3.0mg of a total protein digest starting sample.
Phosphopeptide yields are typically ~ 1-3% of the starting sample tip load and can be determined using the Pierce
Quantitative Colorimetric Peptide Assay Kit (Product # 23290).
 Using a pipette to draw and expel liquid through the spin tip is strongly discouraged and may result in poor, varied
results. Centrifuge Column Adaptors are reusable.
 For optimal results, proceed with the entire procedure in a timely manner and avoid excessive resin drying between steps.
 All solutions should be equilibrated to room temperature prior to enrichment experiment. After each experiment done,
buffer bottle caps should be tightly and securely closed to prevent evaporation prior to storage at 4°C.
.
Procedure for Phosphopeptide Enrichment
A. Materials Required
 High-Select TiO2 Phosphopeptide Enrichment Kit (A32993)
 Collection tubes: Low Protein Binding Microcentrifuge Tubes, 2.0mL (Thermo Scientific, Product # 88379)
 LC-MS Grade Water (Pierce Water, LC-MS Grade, Product # 51140)
 LC-MS Grade 0.1% Formic Acid (Pierce 0.1% Formic Acid (v/v) in Water, LC-MS Grade, Product # 85170)
 Optional: Pierce Quantitative Colorimetric or Fluorometric Peptide Assay (Product # 23275 or 23290 )

B. Suspension of Peptide Sample

 Completely suspend lyophilized peptide sample in 150µL of Solution B. Use vortex mixer with tube stand if necessary.

C. Column Preparation
1. Place a Centrifuge Column Adaptor in a 2.0mL collection tube and insert a TiO2 Spin Tip into the adaptor.
2. Add 20µL of Wash Buffer and centrifuge at 3000 × g for 2 minutes.
3. Add 20µL of Binding/Equilibration Buffer and centrifuge at 3000 × g for 2 minutes.
4. Discard the flow-through. Save the microcentrifuge tube for later Washing Step E1.

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

2
 D. Phosphopeptide Binding
1. Transfer the equilibrated TiO2 Spin Tip and adaptor into a new 2.0 mL microcentrifuge tube.
2. Completely suspend lyophilized peptide sample in 150µL of Binding/Equilibration Buffer. Use vortex mixer with tube
stand if necessary.
Note: Lyophilized peptide samples must be entirely dissolved in Binding/Wash Buffer for optimal results.
3. Apply 150µL of suspended peptide sample to the spin tip. Centrifuge at 1000 × g for 5 minutes.
4. Reapply sample in the microcentrifuge tube to the spin tip. Centrifuge at 1000 × g for 5 minutes. If desired, retain the
flow-through for SMOAC analysis – this fraction is called TiO2-FT.

E. Washing
1. Transfer the TiO2 Spin Tip and adaptor into the collection tube saved from Column Preparation Step C4.
2. Wash column by adding 20µL of Binding/Equilibration Buffer. Centrifuge at 3000 × g for 2 minutes.
3. Wash column by adding 20µL of Wash Buffer. Centrifuge at 3000 × g for 2 minutes. Retain the wash fraction 1.
4. Transfer the TiO2 Spin Tip and adaptor into a new 2.0 mL microcentrifuge tube.
5. Repeat step E2 and E3 one additional time.
6. Wash column by adding 20µL of LC-MS grade water (additional material required). Centrifuge at 3000 × g for 2
minutes. Retain the wash fraction 2.

F. Elution
1. Remove excess liquid by blotting bottom of the spin tip on a clean paper towel (e.g. Kimwipe).
2. Place the spin tip and adaptor in a new collection tube and add 50µL of Phosphopeptide Elution Buffer. Centrifuge at
1000 × g for 5 minutes. Repeat step one additional time.
3. Dry the eluate immediately in a speed vacuum concentrator to remove Phosphopeptide Elution Buffer.
Note: Eluates cannot be stored in Elution Buffer as high pH will lead to loss of phosphates on phosphopeptides.
4. Suspend the eluate with 50µL 0.1% Formic Acid for peptide concentration measurements using the Pierce
Colorimetric Peptide Assay or direct MS analysis.
Note: For < 1mg starting peptide sample amounts, suspend dried elute using 25µL 0.1% Formic Acid.

G. Sample preparation for the SMOAC procedure

1. Combine TiO2-FT from step D4. with the retained wash fraction 1&2 from step E3 & E6..
2. Dry the combined FT-wash fraction by using speed-vac (Note. Do not over-dry. It is normal to see some translucent
jelly-like material at the end of dry step).
3. The dried sample can be stored at -80C until the SMOAC procedure.

Procedure for the SMOAC method

H. Additional Materials Required
 High-Select Fe-NTA Phosphopeptide Enrichment Kit (A32992)
 Collection tubes: Low Protein Binding Microcentrifuge Tubes, 2.0mL (Thermo Scientific, Prodcut # 88379)
 Water, LC-MS Grade (Pierce Water, LC-MS Grade, Product # 51140)
 0.1% Formic Acid, LC-MS Grade (Pierce 0.1% Formic Acid (v/v) in Water, LC-MS Grade, Product # 85170)
 Optional: Pierce Quantitative Colorimetric or Fluorometric Peptide Assay (Product # 23275 or 23290 )

I. Fe-NTA Column Equilibration

1. Remove the bottom closure of the spin column and loosen the screw cap

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

3
 2. Place column in a 2.0mL microcentrifuge collection tube. Centrifuge at 1000 × g for 30 seconds to remove
storage buffer.
3. Remove the screw cap and save it for later Phosphopeptide Binding step J2.
4. Add 200µL of Binding/Wash Buffer. Centrifuge at 1000 × g for 30 seconds and discard the flow-through.
Repeat this step one additional time.
5. Cap the bottom of the column with a white Luer plug. Place the column with the plug into the empty microcentrifuge
tube.

J. Phosphopeptide Binding
1. Completely suspend the dried peptide sample prepared from step G in 200µL of Binding/Wash Buffer by vortexing.
Note: Lyophilized peptide samples must be entirely dissolved in Binding/Wash Buffer for optimal results.
2. Add 200µL of the suspended peptide sample prepared to equilibrated spin column. Close the screw cap.
3. Mix the resin with the sample by holding the screw cap and very gently tapping the bottom plug for 10 sec until the resin is in
suspension. Do NOT vortex nor invert the column to avoid splashing the resin inside the column wall.
Note: Mixing samples by vortexing or inversion results in significantly higher non-specific peptide binding.
4. Incubate for 30 min. Mix the resin gently every 10 min as described in step J2.
5. Carefully remove the bottom plug and the screw cap.
Note: Do not squeeze bottom of the plug during removal as liquid inside the plug can backflow into the spin column.
6. Place the column into the microcentrifuge tube. Centrifuge at 1000 × g for 30 seconds. Discard the flow-through.

K. Washing
1. Wash column by adding 200µL of Binding/Wash Buffer. Centrifuge at 1000 × g for 30 seconds. Repeat this step two
additional times for a total of 3 washes. Discard the flow-through.
2. Wash column by adding 200µL of LC-MS grade water (additional material required). Centrifuge at 1000 × g for 30
seconds.

L. Elution
1. Place column in a new microcentrifuge tube.
2. Add 100µL of Elution Buffer to the column. Centrifuge at 1000 × g for 30 seconds. Repeat this step one additional
time.
Note: The color of resin may turn brown in color, which is normal.
3. Dry the eluate immediately in a speed vacuum concentrator to remove Elution Buffer.
Note: Eluates cannot be stored in Elution Buffer as high pH will lead to loss of phosphates on phosphopeptides.
4. Suspend the dried eluate with 70µL 0.1% Formic Acid for peptide concentration measurements using the
Pierce Colorimetric Peptide Assay or direct LC-MS analysis.
Note: For < 1mg starting peptide sample amounts, suspend dried elute using 40µL 0.1% Formic Acid.

Troubleshooting for Phosphopeptide Enrichment

Problem Possible Cause Solution

No phosphopeptide recovered Phosphatase inhibitors were not used Add phosphatase inhibitors to protein
during protein extraction extraction buffers
Phosphopeptide concentration is too low Increase amount of sample

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

4

Phosphopeptide specificity is low
Spin tip clogged
pH of sample is > 3.5 after suspension
in Binding/Equilibration Buffer
High level of interfering agents in the
sample
Phosphopeptide lost during clean up
LC-MS is not optimal for
phosphopeptide analysis
Non-specific peptides bound to plastics
Washing steps performed in incorrect
order
Particulates in the sample due to
incomplete dissolution of protein digest
sample in Binding/Equilibration Buffer
Desalt protein digest samples before
suspending in Binding/Equilibration
Buffer
Reduce pH < 3 by adding TFA
Modify protein sample preparation to
remove detergents, EDTA, reducing
agent and other interfering substances
Enriched phosphopeptide samples do not
require additional C18 clean up
Avoid trap columns during LC-MS
Check hydrophilic peptide retention on
LC column using Peptide Retention
Time Calibration Mixture (Product #
88320)
Optimize MS methods to avoid or trigger
on phosphopeptide neutral loss
Use low protein binding microcentrifuge
tubes (Product # 88379)
Perform tip equilibration and washing
steps in correct order
Using vortex mixer to completely
dissolve the digest peptide sample
Centrifuge the sample prior to application
to the spin tip

Related Thermo Scientific Products

23275 Pierce Quantitative Colorimetric Peptide Assay
23290 Pierce Quantitative Fluorometric Peptide Assay
28904 Trifluoroacetic Acid, Sequace Grade
51140 Water, LC-MS Grade
51101 Acetonitrile (ACN), LC-MS Grade
A32992 Pierce HiSelect Fe-NTA Phosphopeptide Enrichment Kit
84840 Pierce Mass Spec Sample Prep Kit for Cultured Cells
84868 Pierce High pH Reversed-Phase Peptide Fractionation Kit
88320 Pierce Peptide Retention Time Calibration Mixture
88379 Low Protein Binding Collection Tubes (2.0 mL)
87777 Pierce Detergent Removal Spin Column, 0.5mL, 25 columns
87782 Pierce C18 Tips, 10μL bed, 96 tips
87784 Pierce C18 Tips, 100μL bed, 96 tips
88302 Pierce Graphite Spin Columns, 30 columns
88320 Pierce Peptide Retention Time Calibration Mixture
88328 Pierce HeLa Digest Protein Standard
88811 Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit
89870 Pierce C18 Spin Columns, 25 columns
90057 Pierce™ Trypsin Protease, MS Grade
78427 Halt™ Phosphatase Inhibitor Cocktail

References
1. A versatile mass spectrometry sample preparation procedure for complex protein samples. Antharavally et al. 2013.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-
resource-library/protein-biology-application-notes/mass-spectrometry-sample-preparation-procedure-protein-
samples.html

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax

5
Products are warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product
documentation, specifications and/or accompanying package inserts (“Documentation”). No claim of suitability for use in applications regulated by FDA is made. The warranty
provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment
when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than Buyer. Any model or sample furnished to Buyer is merely
illustrative of the general type and quality of goods and does not represent that any Product will conform to such model or sample.
NO OTHER WARRANTIES, EXPRESS OR IMPLIED, ARE GRANTED, INCLUDING WITHOUT LIMITATION, IMPLIED WARRANTIES OF MERCHANTABILITY,
FITNESS FOR ANY PARTICULAR PURPOSE, OR NON INFRINGEMENT. BUYER’S EXCLUSIVE REMEDY FOR NON-CONFORMING PRODUCTS DURING THE
WARRANTY PERIOD IS LIMITED TO REPAIR, REPLACEMENT OF OR REFUND FOR THE NON-CONFORMING PRODUCT(S) AT SELLER’S SOLE OPTION.
THERE IS NO OBLIGATION TO REPAIR, REPLACE OR REFUND FOR PRODUCTS AS THE RESULT OF (I) ACCIDENT, DISASTER OR EVENT OF FORCE
MAJEURE, (II) MISUSE, FAULT OR NEGLIGENCE OF OR BY BUYER, (III) USE OF THE PRODUCTS IN A MANNER FOR WHICH THEY WERE NOT DESIGNED,
OR (IV) IMPROPER STORAGE AND HANDLING OF THE PRODUCTS.
Unless otherwise expressly stated on the Product or in the documentation accompanying the Product, the Product is intended for research only and is not to be used for any other purpose, including without
limitation, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption by or application to humans or animals.
Current product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.
© 2014 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.

Thermo Fisher Scientific PO Box 117 (815) 968-0747 or (800) 874-3723 thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax