Van Eyk - Methylation Methods

Van Eyk - Methylation Methods
Lysine & Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease
ProteomeXchange: PXD012621
  • Organism: Mus musculus
  • Instrument: Orbitrap Fusion Lumos,TripleTOF 6600
  • SpikeIn: No
  • Keywords: Mass spectrometry, methylation, precursor mass window, DIA-MS, methylome, succinylation, acetylation
  • Submitter: Aaron Robinson
Abstract
Arg and Lys undergo multiple enzymatically driven post-translational modifications (PTMs), including methylation, which may be linked to key cellular processes. Today there is not a method that simultaneously quantifies proteins and the methylome at the site level. We have developed a method that can differentiate an unmodified peptide from its mono-, di- or tri-methylated Arg or Lys counterpart through a data independent acquisition approach that is suitable for both triple TOF and Orbitrap mass spectrometers. This method was further expanded to include Lys acetylation and succinylation, which in conjunction with methylation allow for simultaneous quantification of Lys PTMs in the a single measurement. Our approach leverages small precursor mass windows to physically separate the various methylated species from each other during MS2 acquisition and shows linearity in the quantitation of low abundant peptides. We did this by first creating a biologically hyper-methylated peptide assay library, for mono-, di- and tri-methylated Lys and mono- and di-methylated Arg to which each experimental sample is compared. We further expanded the peptide assay library to include Lys acetylation and succinylation, providing the opportunity to multiplex five Lys and two Arg modification without the need for sample enrichment. To assess its biological relevance, we applied this method to complex liver lysate from differentially methylated in-vivo non-alcoholic steatohepatitis mouse models and show that differential methylation potential and altered acetylation together with total protein changes drive strong novel hypothesis of a regulatory function of post-translational modifications in protein synthesis and mRNA stability. In conclusion: our method, which is suitable for different mass spectrometers along with the corresponding publicly available mouse liver PTM enriched peptide assay library, provides an easily adoptable framework needed for studying global protein methylation, acetylation, and succinylation in any proteomic experiment when total protein quantification is being carried out. Wide adoption of this easy approach will allow more rapid integration of PTM studies and knowledge.
Created on 2/7/19, 11:17 PM
To establish DIA-MS parameters to allow for differentiating a methylated peptide from an unmodified peptide, first a DDA-MS library was built on a Sciex 6600 Triple TOF based on the DDA acquisition of a pool of 400 synthesized peptides (400 femtomoles/peptide) containing either K[Unmodified], K[Monomethyl], K[Dimethyl], or K[Trimethyl]. DIA acquisitions of 400 femtomoles/peptide of the methyl peptide pool were then acquired using a 4 Dalton precursor mass windows and 100 variable mass windows. A DDA library was also created from the methyl peptide pool acquired on the Orbitrap Fusion Lumos (200 femtomoles/peptide). DIA acquisitions of 200 femtomoles/peptide were then acquired with 4, 6, and 12 Dalton fixed precursor mass windows respectively. Each acquisition contains a pool consisting of 100 synthesized unmodified and mono-, di- and tri-methylated peptides. Peptides were resuspended in 0.1% Formic Acid and pooled at 1:1 ratio.

 

 



Clustergrammer Heatmap
 
Download
NV0001_Mouse-Skin_mProphet_Panorama_2024-03-09_19-20-18.sky.zip2024-03-10 20:30:291,6595,7905,79028,90434
XW0008_Cas9Myc_DIAassayLIB_OmBcells_17Nov2023_2024-02-24_08-51-18.sky.zip2024-02-24 12:56:485,20383,67483,675605,04024
XW0009_DIAassayLIB_OmBcells_17Nov2023_2024-02-23_18-35-50.sky.zip2024-02-23 22:06:575,20383,64583,647604,72019
AutoQC-lumos-SysS-MouAD-PFC-C2-B5-B7.sky.zip2024-02-20 07:53:561889414
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B07.sky.zip2024-02-18 11:31:099,778127,624127,624966,34712
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B06.sky.zip2024-02-18 10:45:259,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B05.sky.zip2024-02-18 09:51:569,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B04.sky.zip2024-02-18 01:14:219,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B03.sky.zip2024-02-18 00:22:039,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B02.sky.zip2024-02-17 23:29:529,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B01.sky.zip2024-02-17 18:20:009,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B28.sky.zip2024-02-17 17:30:039,778127,624127,624966,3476
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B27.sky.zip2024-02-17 16:57:559,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B26.sky.zip2024-02-17 15:06:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B25.sky.zip2024-02-17 14:11:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B24.sky.zip2024-02-17 13:17:049,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B23.sky.zip2024-02-17 10:45:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B22.sky.zip2024-02-17 09:52:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B21.sky.zip2024-02-17 09:01:129,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B20.sky.zip2024-02-17 01:24:329,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B19.sky.zip2024-02-17 00:31:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B18.sky.zip2024-02-16 23:42:139,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B17.sky.zip2024-02-16 21:59:109,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B16.sky.zip2024-02-16 21:08:449,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B15.sky.zip2024-02-16 19:45:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B14.sky.zip2024-02-16 18:50:509,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B13.sky.zip2024-02-16 17:05:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B12.sky.zip2024-02-16 16:13:309,778127,624127,624966,34716
XW0008-Myc248_DIAassayLIB_OmBcells_17Nov2023_2024-02-16_10-02-13.sky.zip2024-02-16 15:02:065,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B11.sky.zip2024-02-16 11:03:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B10.sky.zip2024-02-16 10:07:519,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B09.sky.zip2024-02-16 09:14:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B08.sky.zip2024-02-16 08:20:059,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B07.sky.zip2024-02-16 01:08:409,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B06.sky.zip2024-02-16 00:17:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B05.sky.zip2024-02-15 23:29:389,778127,624127,624966,34716
XW0008_nanos3_DIAassayLIB_OmBcells_17Nov2023_2024-02-15_17-02-46.sky.zip2024-02-15 21:13:165,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B04.sky.zip2024-02-15 16:37:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B03.sky.zip2024-02-15 14:42:299,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B02.sky.zip2024-02-15 13:44:359,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B01.sky.zip2024-02-15 12:45:409,778127,624127,624966,34716
AutoQC-lumos-PCs-MouAD-PFC-C2-B5-B7.sky.zip2024-02-14 16:42:502141417344
AutoQC-lumos-PCs-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:42:332141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:42:152141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:42:002141417380
AutoQC-lumos-PCs-MouAD-PFC-C1-B25-B28.sky.zip2024-02-14 16:41:372141417354
AutoQC-lumos-PCs-MouAD-PFC-C1-B21-B24.sky.zip2024-02-14 16:41:002141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 16:40:442141417365
AutoQC-lumos-PCs-MouAD-PFC-C1-B13-B16.sky.zip2024-02-14 16:40:282141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 16:40:082141417347
AutoQC-lumos-SysS-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:10:161889417
AutoQC-lumos-SysS-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:06:251889416
AutoQC-lumos-SysS-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:02:231889422
AutoQC-lumos-SysS-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 15:59:501889418
AutoQC-lumos-SysS-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 14:48:381889410
ZipChip_HR_Metabolomics_2024Protocol_2024-02-05_17-24-05.sky.zip2024-02-05 14:24:28100821594
22AminoAcids_Fully13CLabeled_2024-01-29_14-30-52.sky.zip2024-01-29 11:32:1410444936
RBD_M_Glyco_2024-01-25_15-29-41.sky.zip2024-01-26 17:23:2672923972,3829
20240104_Neg_FMT_MCBAs_isoRemove_Cleaned_Final_2024-01-25_21-40-19.sky.zip2024-01-26 16:43:471010030056
20231220_Neg_FMT_BA_Full_reduce_Res50_High_final_2024-01-04_15-44-59.sky.zip2024-01-26 16:43:47405112176
P179_UNCSet1_ACE_v0p3_2024-01-24_22-42-18.sky.zip2024-01-24 19:51:4423034963724
P179_UNCSet2_ACE_v0p3_2024-01-24_22-37-25.sky.zip2024-01-24 19:40:1117021336726
New_iRBD2024-01-15 23:30:5233474794292
Paired_CSF_Plasma_Serum2024-01-15 23:30:523347479460
Initial_Targeted_Proteomics2024-01-15 23:30:5233474794441
TPAD_VL_CSF_PRTC_APOA1_2024-01-07_23-01-46.sky.zip2024-01-07 23:08:493464642412
TPAD-CSF-SP3_1-5.sky.zip2024-01-05 06:03:432,90823,74323,743189,895396
173_peptides_iRTs_chromatogram_library_2023-12-22_00-47-19.sky.zip2023-12-22 01:06:36311833561,0822
Figure_8B_Freiburg_ALG1-CDG-Patients_Comparison_2023-12-22_02-34-55.sky.zip2023-12-22 01:06:2022691284006
Figures_4_5_6_7_8A_Heidelberg_CDG-Patients_2023-12-22_02-32-43.sky.zip2023-12-22 01:06:20206712439014
Figure_S5_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-59-41.sky.zip2023-12-22 01:06:2021112146
Figure_9_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-53-52.sky.zip2023-12-22 01:06:2021418404
Figures_3_and_S3_HEK_293T_Fibroblasts_HeLa_2023-12-22_01-03-03.sky.zip2023-12-22 01:06:2023701303989
20210301 Calibration Dev_DilutionOil_2023-12-11_10-57-35.sky.zip2023-12-20 00:34:263482654
20210607 Calibration Curve_DilutionDigest_2023-12-11_10-50-40.sky.zip2023-12-20 00:34:2634824108
20210212 Low range exploration 140K-fragmod_Pub_2023-12-08_16-04-13.sky.zip2023-12-20 00:34:263482456
HeatedOilSpike-LowTemp_HighTemp_Combined_Final_2022-05-26_12-00-47.sky.zip2023-12-20 00:34:26591548204
20200715_PeptideSpecificity_SignalRatio_2022-05-25_16-33-02.sky.zip2023-12-20 00:34:2611202212044
20200622_PeptideSpecificityTest_2022-05-25_16-30-20.sky.zip2023-12-20 00:34:2614252713545
20191112_Diff-TempConc_Oil-Spike_24pep_2022-05-25_14-24-35.sky.zip2023-12-20 00:34:2611242715040
20191007_HeatedOilSpike_Extraction_method_24pep_2022-05-25_14-16-21.sky.zip2023-12-20 00:34:2611242715032
20190904_Organic_Aqueous_Extraction_Oil_Spike_24pep_2022-05-25_14-12-26.sky.zip2023-12-20 00:34:2611242715636
September 21 Import V1 (Samples with IS) w Cal Curve_Blanks Deleted_2023-12-01_11-40-59.sky.zip2023-12-02 23:51:3320262687
September 21 Kaylie New Molecule Import v1 (Filtered)_2023-12-01_11-40-01.sky.zip2023-12-02 23:51:3310161698
September 21 Import V1 all samples (Neg mode only)_2023-12-01_11-35-34.sky.zip2023-12-02 23:51:338014514598
THP1_IFN_PRM_Skyline_2023-11-14_14-22-42.sky.zip2023-11-16 13:26:09711771771,5148
CCS_library_v2.sky.zip2023-11-15 14:36:301061,86361,8630
IdentExpression_2023-09-25_14-39-47.sky.zip2023-10-02 20:45:1381581941,16419
2DGel_II_2023-09-25_14-37-39.sky.zip2023-10-02 20:45:13897408805,28034
2DGel_I_2023-09-25_14-36-13.sky.zip2023-10-02 20:45:13252142321,39232
InGelDigest_Der_p_SEA_II_2023-09-18_10-01-52.sky.zip2023-09-21 11:03:445004,0584,42026,52051
InGelDigest_Der_p_SEA_I_2023-09-15_16-14-11.sky.zip2023-09-21 11:03:443272,3822,52115,12652
FASP_Der_p_SEA_2023-09-15_14-24-26.sky.zip2023-09-21 11:03:445504,2964,77128,6261
230504_Myllys_231247ff_2023-09-20_13-22-59.sky.zip2023-09-21 10:31:2410434398
230512MyllysUrea_231247ff_2023-09-21_15-27-13.sky.zip2023-09-21 10:31:24102292
230511Myllys231247ff_AA_Crea_2023-09-20_13-36-24.sky.zip2023-09-21 10:31:242044166126
AutoQC-lumos-SysS-MouAD-C2-B8-10.sky.zip2023-09-17 11:22:321889414
AutoQC-lumos-SysS-MouAD-C2-B5-B7.sky.zip2023-09-17 11:22:301889415
AutoQC-lumos-SysS-MouAD-C2-B1-B4.sky.zip2023-09-17 11:22:281889419
AutoQC-lumos-SysS-MouAD-C1-B9-B12.sky.zip2023-09-17 11:22:261889413

This data is available under the CC BY 4.0 license.