Waters MassPREP Phosphopeptide Enrichment Kit User Manual

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Ma s s P R E P™ P H OSP HO P E P T IDE ENRI C HM ENT K IT

I. INT R O DUC T ION

The MassPREP™ Phosphopeptide Enrichment Kit is a tool for selective

enrichment of phosphopeptides from complex proteomic samples. This

kit contains a micro-scale solid-phase extraction (SPE) plate packed with

a unique affinity sorbent. This affinity SPE does not require pre-loading

the sorbent with metal chelation. Also, superior selectivity is achieved

compared to other existing affinity chromatographic methods.

The selectivity of this sorbent towards phosphopeptides is further

improved by adding a unique chemical to the sample before loading

onto the SPE. This chemical (under the trade name, Enhancer) improves

the overall selectivity by displacing the non-phosphorylated peptides

during the sample loading step.

The MassPREP™ Phosphopeptide Enrichment Kit includes the 96-well

micro elution (µElution) plate, enhancer, and a test sample for method

validation. The test sample is a mixture of four synthetic phosphopeptides

mixed with yeast enolase tryptic peptides in 1:1 molar ratio. A general

guideline for kit operation and specific protocols for phosphopeptide

extraction from complex protein digests are provided. The 96-well

micro elution (µElution) platform conveniently uses a vacuum manifold,

for reproducibility and high throughput of enrichment experiments.

CON T ENT S

I. INT RODUCTION

II. MATERIALS INCLUDED IN THE KIT

III. PHOSPHOPEPTIDE AFFINITY ENRICHMENT METHOD

IV. PHOSPHOPEPTIDE ENRICHMENT KIT USAGE GUIDELINES

Part A. How to Use the MassPREP™ Phosphopeptide Enrichment

μElution Plate

Part B. How to Use the MassPREP™ Enolase/Phosphopeptide

Standard as a Test Sample

Part C. How to Apply Enhancer for Highly Complex Samples

Part D. Sample Preparation for Mass Spectrometry Analysis

V. ORDERING INFORMATION

VI. STORAGE AND DISPOSAL OF USED PLATES

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II. MAT E R I A L S INC L U D E D I N T H E K I T

1. MassPREP™ Phosphopeptide Enrichment µElution plate (part number

186003820). This plate contains 96 wells. Each well is packed

with 2.5 mg of affinity sorbent. The µElution plate is operated by

a vacuum manifold. Related accessories are listed at the end of the

document.

2. MassPREP™ Enhancer (part number 186003821). A total of ten

vials of lyophilized powder are included in the kit (500 mg/vial).

This chemical, termed enhancer, is used to significantly improve the

selectivity of phosphopeptide enrichment by displacing non-phospho-

peptides from the affinity sorbent in the loading step. It is a critical

component of the method.

3. MassPREP™ Enolase/Phosphopeptide Standard (part number 186003286)

contains four synthetic phosphopeptides mixed with yeast enolase

tryptic peptides (Swiss-Prot P00924) in 1:1 molar ratio. The four

synthetic phosphopeptides are modified enolase tryptic peptides that

contain phosphoserine, phosphothreonine, and phosphotyrosine. The

amino acid sequences, phosphorylation sites, and the mass to charge

ratio of the singly and doubly protonated peptide ions are listed

in Table 1. This standard sample can be used as a test sample for

method evaluation before using the customer samples.

III. P H O S P HO P E P T ID E A FF I N I T Y E N RIC H M E NT M E T HO D

Table 2: Solutions Required for the Protocol

Solution Composition of Solution

Condition Solution I Milli-Q® grade water

Condition Solution II 0.2 - 0.5% TFA (v/v) in 80% acetonitrile

Loading Solution (No Enhancer) 0.2 - 0.5% TFA (v/v) in 80% acetonitrile

Loading Solution (with Enhancer) Solubilize the Enhancer with 0.2%-0.5%

TFA in 80% acetonitrile to a final concentration of 50 to 100 mg/mL

Wash Solution I 0.2 - 0.5% TFA in 80% acetonitrile

Wash Solution II Milli-Q® grade water

Elution Solution 100 mM Diammonium Phosphate or

1-2% (v/v) Triethylamine

a. Using highly concentrated enhancer in the sample may result in

loss of phosphopeptides. The recommended concentration is less

than 100 mg/mL.

b. Adding EDTA in the elution solution improves recovery of multiply

phosphorylated peptides, yet interferes with mass spectrometry

analysis. See Section III, D for advices on sample clean up for mass

spectrometry analysis.

Table 1. Amino Acid sequences and m/z ratios of the four synthetic phosphopeptides

Phosphopeptide Description

T18_1P NVPL(pY)K 813.39 407.20

T19_1p HLADL(pS)K 863.40 432.21

T43_1p VNQIG(pT)LSESIK 1368.68 684.84

T43_2P VNQIGTL(pS)E(pS)IK 1448.64 724.83

Sequence [M+H] [M+2H]

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IV. PHOSP HOPE P T IDE ENRICHMENT KI T USAGE GUID ELINES

Part A - How to Use the MassPREP™ Phosphopeptide Enrichment µElution Plate to Condition, Bind, Wash and Elute the Phosphopeptides.

1. Place MassPREP™ Phosphopeptide Enrichment µElution plate on a

vacuum manifold.

2. Wash the wells using 200 µL of Condition Solution I. (vacuum setting:

12 inch Hg).

3. Wash the wells using 200 µL of Condition Solution II. (vacuum setting:

0.5 inch Hg).

4. Prepare the sample by constituting or mixing it with the Loading

Solution. The final volume of the sample should be between 200

to 500 µL.

5. To obtain maximum recovery, use gravity flow, which means vacuum

is turned off. A total of 15 minutes is needed to completely flow

200 µL of the loading solution through a well. Vacuum can be turned

on briefly at the end to force residue liquid through the well.

Part B - How to Use the MassPREP™ Enolase/Phosphopeptide Standard as a Test Sample

1. Solubilize 1 nmol Enolase/Phosphopeptide standard in 1 mL Milli-Q®

water to a final concentration of 1 pmol/µL.

2. Transfer 10 µL of the 1 pmol/ µL standard to 200 µL of the loading

solution: 0.2-0.5% TFA in 80% acetonitrile. Store the remaining

standards at 4 ˚C.

3. Proceed with the MassPREP™ Phosphopeptide Enrichment µElution

protocol to enrich the phosphopeptides (Part A).

4. Reconstitute the lyophilized pellet with 20 µL of Milli-Q® water.

5. Proceed with capillary or nano-LC-MS analysis (see Figure 1).

User can also load the sample directly on the well and turn on the

vacuum manifold to remove the non-phosphorylated peptides. (vacuum

setting: 0.5 inch Hg)

The loading capacity for each well is ~ 100μg of protein digests.

Samples greater than this amount must be split into multiple wells.

6. Wash the well with 200 µL of Wash Solution I, repeat (vacuum setting:

0.5 inch Hg).

7. Wash the well with 200 µL of Wash Solution II (vacuum setting:

7 inch Hg).

8. Elute the phosphopeptides with 100-400 μL of Elution Solution,

repeat. Combine the two elutions (vacuum setting: 7 inch Hg).

9. Neutralize the eluent with pure formic acid

10. Lyophilize the eluent and reconstitute the pellet with a desirable solvent.

Figure 1. NanoLC/MS analysis of: A) Control sample (before afnity enrichment) that is made of 1:1 MassPREP™ Enolase/ Phosphopeptide Standard. B) The four phosphopeptides are selectively enriched using the afnity SPE without using enhancer. The phosphopeptides are marked with symbols T43_1P and T43_2P are observed coeluting. Total sample injected on nanoLC column was about 500 fmol. A trap column is used to remove the excess EDTA in the sample.

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Part C - How to Apply MassPREP™ Enhancer for Highly Complex Samples

MassPREP™ Enhancer can be used to improve the overall selectivity

of the phosphopeptides from a highly complex biological sample (e.g.,

whole cell lysate digested with proteolytic enzymes).

1. Weigh 100 mg Enhancer from the vial and transfer to a separate

vial. Keep the rest of Enhancer in the capped vial for later use, and

store the vial at room temperature. Expiration time of the Enhancer

powder is ~ 3 years; storage is at room temperature.

2. Prepare 100 mg/mL Enhancer solution by adding 1 mL of the loading

solution to the weighed-out enhancer, vortex gently. (Note: The

expiration time for this reconstituted solution is ~ 1 week; storage

is at 4 ˚C)

3. Prepare the peptide sample by solubilizing it with 200 µL of the

Enhancer solution.

4. Precede with Part I to isolate the phosphopeptides.

V. ORD E R I N G INFORMAT ION

Table 2. MassPREP™ Phosphopeptide Enrichment Kit Ordering Information

Description Part Number Units/ki Amt/Unit

MassPREP™ Phosphopeptide Enrichment Kit 186003864 – –

MassPREP™ Phosphopeptide Enrichment µElution Plate

MassPREP™ Enolase/Phosphopeptide Standard

MassPREP™ Enhancer™ 186003821 Qty 10 500mg

Table 3. Vacuum Manifold Accessories Ordering Information

SPE Vacuum Pump (115, 60 Hz) 725000417

SPE Vacuum Pump (240, 50 Hz) 725000418

µElution Plate Manifold 186001831

Manifold Waste Tray, disposable, 25/pkg WAT058942

350 µL Sample Collection Plate, polypropylene 96 wells, 50 plates/pkg

186003820 Qty 1 1 plate

186003286 Qty 1 1 nmole

WAT058943

Part D - Sample Preparation for Mass Spectrometry Analysis

The phosphopeptides enriched using this kit, will be subjected to Mass

Spectrometry analysis. Here are some advices for post-kit sample

preparation.

1. For MALDI-MS analysis, use a reversed-phase cartridge (e.g, ZipTip

to remove Diammonium Phosphate or other potential contaminants

from the phosphopeptides.

2. For capillary or nano-scale LC/MS analysis, a reversed-phase trap

column is needed to remove excess EDTA, Diammonium Phosphate

and other contaminants from the sample.

™

V I . S T O R AG E A N D D I S P O S A L O F U S ED P L AT E S

Cartridges stored in their original sealed pouch remain stable for long

periods. To store unused cartridges in opened pouches, squeeze the

air out of the pouch, fold over the open end of the pouch twice, seal

with tape and store in a dessicator. Contaminants that may interfere

with the effectiveness of the affinity sorbent are high quantities of

)

detergents and organic salts, such as urea. Dispose off used cartridges

and plates safely in accordance with applicable government or local

regulations.

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Waters and MassPREP are trademarks of Waters Corporation. Milli-Q and ZipTip are registered trademarks of Millipore Corporation. Swiss-Prot is a trademark of Institut Suisse de Bioinformatique Foundation Switzerland.

Waters Corporation

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Waters MassPREP Phosphopeptide Enrichment Kit User Manual

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Frequently Asked Questions

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