Oct 24, 2023

Public workspaceIn vitro LRRK2 kinase activity assay using mass-spectrometry as readout V.2

DOI
dx.doi.org/10.17504/protocols.io.6qpvr385ovmk/v2
  • 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany;
  • 2Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany
Verena Dederer
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr385ovmk/v2
Protocol Citation: Verena Dederer, Deep Chatterjee, Sebastian Mathea, Stefan Knapp 2023. In vitro LRRK2 kinase activity assay using mass-spectrometry as readout. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr385ovmk/v2Version created by Verena Dederer
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 07, 2023
Last Modified: October 24, 2023
Protocol Integer ID: 87485
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000519
Abstract
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine in vitro substrate phosphorylation for any purified kinase substrate pair.
It can be used to determine inhibition curves by addition of concentration series of kinase inhibitors.

Reaction is carried out in 50 µL reaction mix containing final concentration of 50 nM kinase and 5 µM substrate.
Materials
Purified proteins

kinase: LRRK2RCKW
protein sequence: KKAVPYNRMKLMIVGNTGSGKTTLLQQLMKTKKSDLGMQSATVGIDVKDWPIQIRDKRKRDLVLNVWDFAGREEFYSTHPHFMTQRALYLAVYDLSKGQAEVDAMKPWLFNIKARASSSPVILVGTHLDVSDEKQRKACMSKITKELLNKRGFPAIRDYHFVNATEESDALAKLRKTIINESLNFKIRDQLVVGQLIPDCYVELEKIILSERKNVPIEFPVIDRKRLLQLVRENQLQLDENELPHAVHFLNESGVLLHFQDPALQLSDLYFVEPKWLCKIMAQILTVKVEGCPKHPKGIISRRDVEKFLSKKRKFPKNYMSQYFKLLEKFQIALPIGEEYLLVPSSLSDHRPVIELPHCENSEIIIRLYEMPYFPMGFWSRLINRLLEISPYMLSGRERALRPNRMYWRQGIYLNWSPEAYCLVGSEVLDNHPESFLKITVPSCRKGCILLGQVVDHIDSLMEEWFPGLLEIDICGEGETLLKKWALYSFNDGEEHQKILLDDLMKKAEEGDLLVNPDQPRLTIPISQIAPDLILADLPRNIMLNNDELEFEQAPEFLLGDGSFGSVYRAAYEGEEVAVKIFNKHTSLRLLRQELVVLCHLHHPSLISLLAAGIRPRMLVMELASKGSLDRLLQQDKASLTRTLQHRIALHVADGLRYLHSAMIIYRDLKPHNVLLFTLYPNAAIIAKIADYGIAQYCCRMGIKTSEGTPGFRAPEVARGNVIYNQQADVYSFGLLLYDILTTGGRIVEGLKFPNEFDELEIQGKLPDPVKEYGCAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQLSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVTCLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCLSESTNSTERNVMWGGCGTKIFSFSNDFTIQKLIETRTSQLFSYAAFSDSNIITVVVDTALYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKENKESKHKMSYSGRVKTLCLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQKEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE

substrate: Rab8A
protein sequence: GHMDYLFKLLLIGDSGVGKTCVLFRFSEDAFNSTFISTIGIDFKIRTIELDGKRIKLQIWDTAGQERFRTITTAYYRGAMGIMLVYDITNEKSFDNIRNWIRNIEEHASADVEKMILGNKCDVNDKRQVSKERGEKLALDYGIKFMETSAKANINVENAFFTLARDIKAKMDKK

Buffers and Reagents

reaction buffer: 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2

ATP 100 mM stock in dH2O

mass spec buffer: dH2O + 0.1% formic acid
Step-by-Step Protocol
Step-by-Step Protocol
Prepare 50 µL reaction mix I: 50 nM purified LRRK2RCKW and 5 µM Rab8 substrate in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.
Aliquot 25 µL per tube.


Note
If you want to run multiple reactions prepare a master mix and aliquot 25 µL per tube.

Prepare 25 µL reaction mix II: 2 mM ATP + 2.5 mM MgCl2 in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.

Note
Multiply if you want to run more than one reaction.

Prepare 25 µL reaction mix III: same as reaction mix II but no ATP as negative control.
Start reaction by adding 25 µL reaction II to reaction mix I.
Note: for negative control add 25 µL reaction mix III to second aliquot of reaction mix I.
Mix by vortexing and brief centrifugation for 30 sec with 500xg.
Incubate reaction for 3 h at room temperature.
Note: depeding on kinase this may be shorter or longer.
Stopp reaction by adding 50 µL mass spec buffer (dH2O+0.1% formic acid).
Store at -80°C or proceed directly with mass spectrometry analysis.




Note
For determination of inhibition curves, prepare a concentration series of inhibitor in DMSO. Prepare a master mix of kinase and substrate pair and aliquot 25 µL per tube. Add the desired amount of inhibitor to each of the tube and incubate all reactions at room temperature.

Note
For analysis: substrate phosphorylation/turnover can be determined as ratio between phosphorylated and unphosphorylated peak intensity for each sample analyzed.