Thermo Fisher Scientific Phospho-enrichable Crosslinkers User guide

Brand: Thermo Fisher Scientific

Model: Phospho-enrichable Crosslinkers

Type: User guide

Phospho-enrichable Crosslinkers

Catalog Numbers A52286 and A52287

Doc. Part No. 2162755 Pub. No. MAN0025824 Rev. A.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and

gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/support.

Product description

Disuccinimidyl Phenyl Phosphonic Acid (DSPP or PhoX) and tert-Butyl Disuccinimidyl Phenyl Phosphonate (TBDSPP or tBu-PhoX) are phospho-

enrichable crosslinkers containing an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of a 7-atom spacer arm and a phosphonic

acid3 or phosphonate ester (tBu-PhoX). DSPP and TBDSPP react eciently with primary amine groups (-NH2) in pH 7-9 buers to form stable

amide bonds and contain phospho groups that can be used to enrich crosslinked peptides using immobilized metal anity chromatography

(IMAC) or metal oxide anity chromatography (MOAC). The phosphonic acid group of PhoX and tBu-PhoX (after acid cleavage) is not a substrate

for phosphatases, which can be used to remove endogenous phosphopeptides prior to enrichment.

Chemical crosslinking in combination with MS is a powerful method to determine protein structure and protein-protein interactions 1,2. This

method has been applied to recombinant and native protein complexes and to whole cell lysates or intact unicellular organisms in eorts to

identify protein-protein interactions on a global scale. Both traditional non-cleavable and MS-cleavable crosslinkers can be used for identiﬁcation

of protein-protein interaction sites, but phospho-enrichable crosslinkers are advantageous because they can be used to enrich low-abundant

crosslinked peptides and thereby improve MS identiﬁcation rates.

Contents and storage

Contents Cat. No. Amount Storage

DSPP (Disuccinimidyl Phenyl Phosphonic Acid, PhoX)

MW = 440.25 Spacer Arm = 4.8 Å

A52286

50 mg Store at –20°C protected from

moisture.

TBDSPP (tert-Butyl Disuccinimidyl Phenyl Phosphonate, tBu-PhoX)

MW = 552.46 Spacer Arm = 4.8 Å

A52287

Additional information

• Crosslinkers are moisture sensitive. To avoid moisture condensation on the product, equilibrate vial to room temperature before opening.

Prepare these crosslinkers immediately before use. The NHS-ester moiety readily hydrolyzes to become non-reactive; therefore, do not

prepare stock solutions for storage. Discard any unused reconstituted crosslinker.

• Hydrolysis of the NHS ester competes with crosslinking primary amines of proteins/peptides (acylation). Acylation is favored near neutral pH

(pH 6–9) and with concentrated protein solutions.

• Use amine-free buers at pH 7–9 such as PBS (100 mM sodium phosphate, 150 mM NaCl, pH 7.4, Cat. No. 28372), 20 mM HEPES, 100 mM

carbonate/bicarbonate, or 50 mM borate. Buers containing primary amines such as Tris or glycine compete with acylation.

• Dissolve DSPP and TBDSPP in a dry, water-miscible organic solvent, such as DMSO, before diluting to <10% solvent in ﬁnal aqueous

reaction buer. We recommend ﬁrst dissolving DSPP in acetonitrile and drying using a speedvac before reconstitution in DMSO for

maximal crosslinker solubility. DSPP is soluble in aqueous buers; however, TBDSPP may precipitate if added directly to aqueous buer

at concentrations >30 mM.

• The protection groups of TBDSPP must be removed using TFA to liberate the phosphonic acid group prior to phosphoenrichment.

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

• Excess, unreacted DSPP and TBDSPP must be removed before enrichment of crosslinked peptides. Acetone or methanol/chloroform

precipitation is recommended for protein-level clean up. C18 and peptide desalting columns are recommended for crosslinked peptide clean

up. EasyPep™ MS sample prep peptide clean up columns/plates are not recommended for clean up of crosslinked peptides.

Required materials not supplied

• Phosphopeptide enrichment kit (Cat. No. A32993, A32992, A52283, or A52284)

• Nuclease (e.g., Pierce™ Universal Nuclease for Cell Lysis, Cat. No. 88700), probe sonicator or equivalent

• Protein assay kit (e.g., Pierce™ BCA Protein Assay Kit, Cat. No. 23227)

• Peptide assay kit (e.g., Pierce™ Quantitative Fluorometric Peptide Assay, Cat. No. 23290)

• Phosphatase (e.g., Pierce™ Alkaline Phosphatase, Cat. No. 31392)

• 10K or 30K MWCO ﬁlters (Pierce™ Protein Concentrators, PES, 30K MWCO, 0.5 mL, Cat. No. 88513) or equivalent

• Trypsin and LysC MS-grade proteases (Cat. No. 90057 or A41007)

• C18 tips or peptide desalting columns (e.g., Cat. No. 89851)

Crosslink in vitro proteins

Both DSPP (PhoX) and TBDSPP (tBu-PhoX) can be used for crosslinking proteins in vitro. This protocol is designed to result in sucient

crosslinking to facilitate subsequent MS analysis, but not disturb the tertiary structure of the protein from excessive crosslinking. Maintaining a

protein concentration in the micromolar range during the reaction reduces unwanted intermolecular crosslinking between proteins.

1. Dissolve the protein in 490 µL of 20 mM HEPES buer (pH 7.5) at 10 µM.

2. For DSPP, prepare a 50 mM stock solution of crosslinker by dissolving 1 mg DSPP in 45.4 µL of water. For TBDSPP, prepare a 50 mM stock

solution of crosslinker by dissolving 1 mg of TBDSPP in 36.4 µL of DMSO or acetonitrile. DMSO is optimal when the crosslinker is prepared

in MeCN and then dried down.

3. Add 10 µL of crosslinker solution to the protein for a ﬁnal concentration of 1 mM (100-fold molar excess of crosslinker over the protein

concentration) and incubate at room temperature for 1 hour.

Note: Titrate the ﬁnal crosslinker concentration to determine the optimal molar excess for crosslinking. Typically, a minimal 20-fold molar

excess is required to observe crosslinking, but up to 300-fold may be necessary to fully crosslink some protein complexes.

4. Terminate the reaction by adding 1 M NH4HCO3 or Tris buer to a ﬁnal concentration of 20 mM to each sample.

5. Proceed to “Prepare crosslinked protein samples for MS” on page 2.

Crosslink intra- and extra-cellular proteins

TBDSPP (tBu-PhoX) crosslinking can be performed on cells in suspension or on adherent cells in culture plates. In the latter situation, diusion of

the crosslinking reagent to all cell surfaces will be limited and will occur predominantly on the exposed surface. Culture media must be washed

from the cells; otherwise, amine-containing components will quench the reaction. Using a more concentrated cell suspension is most eective as

less reagent is required in the reaction.

1. Remove media from 107 cells and wash twice with ice-cold PBS.

2. Prepare a 50 mM stock solution of TBDSPP by dissolving 5 mg in 182 µL of DMSO.

3. Add a ﬁnal crosslinker concentration (diluted in PBS) of 1-5 mM to the cells.

4. Incubate the reaction at room temperature for 10 minutes or on ice for 30 minutes.

5. Stop the reaction by adding 1 M NH4HCO3 or Tris Buer to a ﬁnal concentration of 20 mM and incubating for 15 minutes.

6. Rinse cells twice with PBS to remove excess non-reacted crosslinker from cells.

7. Harvest cells and add 5 pellet-cell volumes of EasyPep™ Lysis Buer (Cat. No. A45735) to extract crosslinked proteins.

Note: Addition of universal nuclease or sonication is recommended to reduce lysate viscosity.

8. Add 20 µL of alkaline phosphatase per 500 µg of cell lysate and incubate in a thermomixer at 37°C at 500 rpm for 30 minutes.

9. Proceed to “Prepare crosslinked protein samples for MS” on page 2.

Prepare crosslinked protein samples for MS

It is recommended to evaluate the extent of crosslinking by 1-D SDS-PAGE. Stain gels with MS-compatible stains such as GelCode™ Blue

Stain Reagent (Cat. No. 24590), Imperial™ Protein Stain (Cat. No. 24615) or MS-compatible silver stains such as SilverSNAP™ Stain for Mass

Spectrometry (Cat. No. 24600). The occurrence of crosslinking at this stage establishes the optimal condition for the crosslinking reagents.

1. For crosslinked proteins, remove excess crosslinker using PES 10K or 30K MWCO ﬁlters (Cat. No. 88502 or equivalent). For crosslinked

cells/lysates, perform acetone precipitation or equivalent protein-level cleanup.

2. Reduce and alkylate the sample with TCEP and 2-chloroacetamide to a ﬁnal concentration in solution of 10 mM and 20 mM, respectively.

Incubate the samples at 95°C for 10 minutes, then cool samples for 5 minutes at 4°C.

3. Add LysC to 1:100 and Trypsin to 1:50 (µg enzyme:µg protein) and incubate overnight (>16 hours) at 37°C in a thermomixer set at 500 rpm.

4. Acidify samples with TFA to stop the reaction and prepare samples for cleanup.

Note: For TBDSPP crosslinked samples, add 2% TFA and incubate for 1 hour at 37°C in a thermomixer set at 500 rpm to deprotect the

crosslinker prior to phospho-enrichment.

5. 5. Clean up peptides using C18 tips or peptide desalting spin columns (Cat. No. 89851).

2Phospho-enrichable Crosslinkers User Guide

Enrich crosslinked peptides

This protocol is designed to enrich 200 µg of DSPP or TBDSPP crosslinked peptides using the High-Select™ Fe-NTA Magnetic Phosphopeptide

Enrichment Kit (Cat. No. A52283). Enrichment of 100-1,000 µg of peptide can be performed with bead and buer volumes scaled accordingly.

Clean peptide samples should be lyophilized using a speedvac prior to enrichment.

1. Dissolve 200 µg of clean peptide using 200 µL of Binding/Wash Buer in a low protein-binding tube.

2. Transfer 20 µL of the magnetic Fe-NTA bead slurry using a cut or wide bore pipet tip to a new microcentrifuge tube and remove the storage

buer using a magnetic stand.

3. Wash the resin by adding 40 µL of Binding/Wash Buer, then vortex brieﬂy and remove the buer using a magnetic stand.

4. Repeat the wash once for a total of 2 washes.

5. Add 20 µL of the Binding/Wash Buer to the resin and transfer slurry to the peptide solution (1:10, µL bead slurry:µg sample).

6. Incubate at room temperature for 30 minutes with end-over-end mixing.

7. Collect the beads using a magnetic stand and remove the unbound peptide solution.

8. Wash the resin by adding 40 µL of Binding/Wash Buer, then vortex brieﬂy and remove the buer using a magnetic stand.

9. Repeat the wash twice for a total of three washes.

10. Wash the resin by adding using 40 µL of MS-grade water, then vortex brieﬂy and remove the water using a magnetic stand.

11. Add 40 µL of Phosphopeptide Elution Buer, then vortex brieﬂy and incubate for 1 minute at room temperature.

12. Repeat elution for a total of 2 elutions.

13. Combine the eluted phosphopeptide samples, transfer to a low protein-binding tube, and speedvac dry before LC-MS analysis.

We recommend centrifugation of the eluted peptide samples at 10,000 × g for 1 minute before transferring supernatant to avoid bead

carryover before drying. Use of C18 tips or trap columns are also recommended to ensure resin particles do not interfere with LC-MS

analysis.

14. Resuspend dried phosphopeptide sample using 0.1% FA for LC-MS analysis.

Analyze crosslinked proteins using mass spectrometry

1. Separate and analyze the peptide mixture containing crosslinked products by liquid chromatography-mass spectrometry (LC-MS).

2. Detect the resulting inter- and intra-molecularly crosslinked peptides in the mass spectra by their modiﬁed peptide mass dierence.

Note: Peptides have mass shifts in both their fully crosslinked (Mip, Mi) (Table 1) and partially hydrolyzed forms (MOH, MNH2, MTris) (Table 2).

Table 1 Mass modiﬁcations associated with crosslinked peptides.

Reagent Modiﬁcation Inter-peptide crosslink (Mip) Intra-peptide crosslink (Mi)

DSPP, TBDSPP C8 H3 O5 P 209.97181 amu 209.97181 amu

Table 2 Mass modiﬁcations associated with crosslinker monoadducts.

Reagent Monolink water quench (MOH) Monolink ammonia quench (MNH2) Monolink Tris quench (MTris)

DSPP, TBDSPP 227.98237 amu 226.99836 amu 331.04570 amu

Analyze crosslinked protein data

Commercially available or free bioinformatics software can analyze complex mixtures of crosslinked peptides. Public domain packages include:

• ExPASy Proteomics Tool in the Swiss-Prot Database 'FindPept' at expasy.org

• Protein Prospector at prospector.ucsf.edu/prospector

• Plink at http://pﬁnd.ict.ac.cn/software/pLink

• XlinkX at xlinkx.hecklab.com is available as a node in Thermo Scientiﬁc™ Proteome Discoverer™ 2.2 Software.

Related products

Product Cat. no.

Iodoacetamido-LC-Phosphonic Acid (6C-CysPAT) A52285

Disuccinimidyl sulfoxide (DSSO) A33545

Disuccinimidyl dibutyric acid (DSBU) A35459

Pierce™ BCA Protein Assay Kit 23225

Pierce™ Rapid Gold BCA Protein Assay Kit A53225

Pierce™ Quantitative Colorimetric Peptide Assay Kit 23275

Pierce™ Quantitative Fluorometric Peptide Assay 23290

Pierce™ Universal Nuclease for Cell Lysis 88700

Pierce™ Trypsin/Lys-C Protease Mix, MS Grade A40007

Pierce™ CIP Alkaline Phosphatase 31391

Pierce™ Peptide Desalting Spin Columns 89851

High-Select™ TiO2 Phosphopeptide Enrichment Kit A32993

High-Select™ Fe-NTA Phosphopeptide Enrichment Kit A32992

High-Select™ Fe-NTA Magnetic Phosphopeptide Enrichment Kit A52283

High-Select™ Fe-NTA Magnetic Agarose A52284

Phospho-enrichable Crosslinkers User Guide 3

References

1. Arlt, C., et al. 2016. Integrated workﬂow for structural proteomics studies based on crosslinking/mass spectrometry with an MS/MS-cleavable

crosslinker. Anal Chem, 88(16):7930-7.

2. Liu, F., et al. 2015. Proteome-wide proﬁling of protein assemblies by crosslinking mass spectrometry. Nat Methods, 12(12):1179-84.

3. Steigenberger, B., et al. 2019. PhoX: An IMAC-Enrichable Cross-Linking Reagent. ACS Cent. Sci. 5(9):1514-22

Troubleshooting

Observation Possible cause Recommended action

Minimal or no crosslinking observed. NHS ester hydrolyzed. Allow product to equilibrate to room temperature before opening.

Ensure DMSO used to prepare stock solution is dry (e.g., anhydrous

molecular sieves).

Used inappropriate buer for

crosslinking.

Avoid buers that contain primary amines such as Tris or glycine. Ensure

buer pH is 7–8.

Used inappropriate molar excess of

product to target.

Optimize product-to-target ratio. Use 20–400 molar excess for 2 mg/mL

protein or 10–15 molar excess for 10 mg/mL protein.

Lysines were not within optimal

crosslinking distance.

Use a crosslinker with a dierent linker length or reactivity (e.g., sulfhydryl

or carboxylic acid).

Poor solubility of crosslinker in stock

solutions.

Dissolve DSPP and TBDSPP using dry DMSO. Alternatively, DSPP can

ﬁrst be dissolved in acetonitrile and speedvac dried before reconstitution in

DMSO. DSPP is also soluble in water.

Poor enrichment of crosslinked

peptides

Incomplete removal of excess

crosslinker.

Perform protein-level cleanup prior to digestion.

Incomplete hydrolysis of tBu groups of

TBDSPP.

Increase amount of TFA, incubation time, or incubation temperature for

acid cleavage prior to cleanup and enrichment.

Competition from endogenous

phosphopeptides.

Dephosphorylate phosphopeptides using alkaline phosphatase.

Low phosphopeptide speciﬁcity. Refer to troubleshooting in the appropriate phosphopeptide enrichment

kit.

Limited product warranty

Life Technologies Corporation and/or its aliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of

Sale at www.thermoﬁsher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at

www.thermoﬁsher.com/support.

Thermo Fisher Scientiﬁc | 3747 N. Meridian Road | Rockford, Illinois 61101 USA

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0025824

Revision Date Description

A.0 18 November 2021 New manual for Phospho-enrichable Crosslinkers.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

applicable Limited Use Label Licenses.

thermoﬁsher.com/support | thermoﬁsher.com/askaquestion

thermoﬁsher.com

18 November 2021

Thermo Fisher Scientific Phospho-enrichable Crosslinkers User guide

Brand: Thermo Fisher Scientific

Model: Phospho-enrichable Crosslinkers

Type: User guide

Download PDF

report

Thermo Fisher Scientific Phospho-enrichable Crosslinkers User guide

Thermo Fisher Scientific Phospho-enrichable Crosslinkers User guide

Related papers

Other documents