Identification of Post Translational Modifications Beatrix Ueberheide March 25th 2019

Homework from last lecture

Assigned Paper

Ubiquitination - MS

Jackson Paper: USP4 ubiquitination Catalytic dead USP4 (Cys311A) no longer interacts with CtIP and MRN Could it be that ubiquitination of USP4 is blocking the interaction with CtIP and MRN and that inactive USP4 can no longer de-ubiquitinates itself and hence prevents interaction? Co-expressed USP4 with HA tagged ubiquitin: GFP-USP4 WT; GFP-USP4 CD; GFP Performed affinity purification using the HA tag and Western Blot using the GFP handle Performed GFP affinity purification followed by LC-MS Found a sh%^$ load of ubiquitin

Table S4

Sites of ubiquitination on USP4 CD WT STLVCPECAK 1164.5388 [M+1H]+1

Ubiquitinated spectrum

Which cysteine is ubiquitinylated?

Let’s check public databases: www.thegpm.org

From The GPM

From The GPM

Could it be this?

Easy Problem to have avoided

Figure Legend

Figure Legend

Figure Legend

Figure Legend

Characterizing PTMs

Detecting PTMs In theory quite simple, only the mass of the amino acid changes In practice difficult for several reasons Peptide is too short or too long to be detected Not enough sequence coverage to assign the site of modification Modification is present only in a small percentage Peptide is never selected for MS/MS Several amino acids in the peptide could be modified Modification might interfere with fragmentation Not enough sequence coverage to identify the peptide

Biological Mass Spectrometry Proteolytic digestion Protein(s) Peptides Base Peak Chromatogram MS 500 1000 1500 m/z Time (min) Mass Spectrometer 200 600 1000 m/z MS/MS Database Search Manual Interpretation

Searching Proteomics Data GSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTP Protein Digestion LFTFHADICTLPDTEK 1850.8993 RPCFSALTPDETYVPK 1823.8906 MPCTEDYLSLILNR 1667.8131 VPQVSTPTLVEVSR 1511.8427 DDPHACYSTVFDK 1497.6314 Peptide Mass Measurement 500 1000 1500 m/z 1850.8993 1850.8906 1850.8805 1850.8914 1850.8868 924.9537 MS Peptide Fragmentation 200 600 1000 m/z MS/MS

Proteomics is more than Mass Spec Enrichment Fractionation Sample Protein Extraction Protein Digestion Data Analysis LC-MS Peptide Clean up Enrichment Fractionation

Inherent difficulty of proteome characterization Proteolytic digestion Protein(s) Peptides Size of peptides depends entirely on the protein sequence (K, R, D, E, Y, F,….) Peptides too short or too long are not detectable If those peptides carry PTMs, they go undetected as well

Inherent difficulty of proteome characterization Proteolytic digestion Protein(s) Peptides Protein A Protein B Protein C Protein D Protein E One protein group is reported!

Inherent difficulty of proteome characterization Proteolytic digestion Protein(s) Peptides Protein A Cancer Protein A Healthy

The Central Dogma Proteomics Metabolomics seconds to minutes hours (seconds) Genomics Transcriptomics Proteomics Metabolomics Adapted from Patti et al (2011) Nature Reviews| Molecular Cell Biology

Proteomics

Proteomics Protein Production/Changes Protein PTM modification/Changes Protein-Protein Interaction/Changes Protein Structural Changes

Dynamic Range 200ng HeLa lysate: ~2500 proteins >8000 proteins 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) Relative Abundance 444.74 477.31 408.73 492.79 652.36 472.77 425.72 599.76 416.25 590.81 566.77 567.78 420.79 533.32 644.73 757.40 895.95 446.91 655.85 584.80 680.37 533.27 726.05 911.40 455.32 899.46 200ng HeLa lysate: ~2500 proteins >8000 proteins 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Time (min) Relative Abundance 575.31 395.24 476.23 671.82 682.70 480.79 492.75 467.26 518.21 547.32 663.58 829.38 489.96 371.10 518.03 637.98 749.80 669.59 756.43 992.46 623.58 593.83 840.41 738.06 871.38 200ng Human Plasma: ~250 proteins

Success Rate of a Proteomics Experiment Distribution of Protein Amounts Number of Proteins Fractionation Enrichment Proteins Detected Log (Protein Amount) DEFINITION: The success rate of a proteomics experiment is defined as the number of proteins detected divided by the total number of proteins in the proteome. J. Eriksson, D. Fenyö, "Improving the success rate of proteome analysis by modeling protein-abundance distributions and experimental designs". Nature Biotechnology 25 (2007) 651-655.

Fractionation and Enrichment One sample becomes 10 or more Fractions Protein identification increases from ~2000 to ~8000 ( 2 hours versus 60 hours instrument time)

Fractionation and Enrichment increases the MS instrument time

Multiplexing for Quantitation SILAC TANDEM MASS TAGS

SILAC

Multiplex Analysis (TMT)

iTRAQ

Multiplex Analysis (TMT)

TMT (iTRAQ)

MS2 of a peptide Peptide: AVDTWSWGER Protein: Galectin 3 binding protein May stimulate host defense against viruses and tumor cells

Zoom in

Quantitative multiplexed proteomic analysis of TNKS DKO cells 7,254 proteins quantified: 608 showed significant change in abundance 287 increased in the DKO 74 of the 287 contained a TNKS-binding site 23 of the 74 tankyrase targets are shown in the heatmap Highlighted proteins were selected for validation Bhardwaj A., Yang, Y., Ueberheide, B., Smith, S., Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation, Nature Communications, 8:2214 (2017)

Characterizing PTMs

Modified Peptide is not abundant enough to be selected for MS/MS enriching for the modification Phospho specific enrichment (IMAC, TiO2) Antibody based enrichment (ubiquitin, phosphotyrosine)

Fractionation and Enrichment Total Cell Lysate 1824 Protein Groups 12389 Peptides 26 phosphorylated peptides 100 % S & T 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 Time (min) 5 10 15 Relative Abundance Total cell lysate 1 HILIC Fraction 917 Protein Groups 1208 Peptides 95 phosphorylated peptides 100 % S & T 0 % Y 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Time (min) 5 10 15 60 65 70 75 80 85 90 95 100 Relative Abundance

Fractionation and Enrichment 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Time (min) 5 10 15 60 65 70 75 80 85 90 95 100 Relative Abundance Total cell lysate 1 HILIC Fraction 917 Protein Groups 1208 Peptides 95 phosphorylated peptides 100 % S & T 0 % Y Total Cell Lysate 1 HILIC Fraction TiO2 Enrichment 347 Protein Groups 419 Peptides 387 phosphorylated peptides 99.2 % S & T 0.8 % Y 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 Time (min) 5 10 15 25 35 45 55 65 70 75 80 85 90 95 100 Relative Abundance

Enrichment for pY Pre-Enrichment Post-Enrichment NL: 2.44E9 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 Time (min) Relative Abundance 477.3055 472.7690 416.2502 488.7277 442.5899 445.9025 652.3596 872.4121 590.8138 569.2763 796.9095 516.8010 489.6061 644.7284 566.7671 714.3454 452.2126 526.9341 499.7473 895.9489 600.2489 880.4102 584.8013 911.4047 933.7854 406.3527 470.9005 498.8081 989.7180 537.8790 939.4348 450.3788 434.3840 419.1702 900.5027 995.0111 445.1200 633.2947 840.0427 406.3518 681.2819 714.3449 476.3057 453.3427 432.2793 520.3320 575.7687 493.5727 608.3846 563.5903 475.8850 644.7281 516.8008 564.3422 445.1194 445.1195 567.9413 796.9098 489.3137 487.6485 511.3267 516.9537 450.3782 599.3796 445.1192 445.1191 445.1189 NL: 2.44E9 NL: 2.50E7 Pre-Enrichment Post-Enrichment

Enrichment for pY Pre-Enrichment Post-Enrichment 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 Time (min) Relative Abundance 477.3055 472.7690 416.2502 488.7277 442.5899 445.9025 652.3596 872.4121 590.8138 569.2763 796.9095 516.8010 489.6061 644.7284 566.7671 714.3454 452.2126 526.9341 499.7473 895.9489 600.2489 880.4102 584.8013 911.4047 933.7854 406.3527 470.9005 498.8081 989.7180 537.8790 939.4348 450.3788 434.3840 419.1702 900.5027 995.0111 445.1200 633.2947 840.0427 406.3518 681.2819 714.3449 476.3057 453.3427 432.2793 520.3320 575.7687 493.5727 608.3846 563.5903 475.8850 644.7281 516.8008 564.3422 445.1194 445.1195 567.9413 796.9098 489.3137 487.6485 511.3267 516.9537 450.3782 599.3796 445.1192 445.1191 445.1189 NL: 2.44E9 NL: 2.50E7 Pre-Enrichment   Total number of Proteins Total number of Peptides Total Phospho Peptides Tyrosine Phospho Peptides Pre-Enrichment 2364 18826 221 5 Post-Enrichment 988 2919 583 574 Post-Enrichment

Enrichment Strategies for the Detection of Phosphorylated Peptides Unphosphorylated single phosphorylation multiple phosphorylation Hydrophilic Interaction Chromatography (HILIC) Phosphopeptides elute later than their unphosphorylated counterparts Stationary phase is hydrophilic Mobile phase is hydrophobic Peptide elute when it is more soluble in the solvent, the higher the charge the less soluble in the organic phase Works for PO4 but other modifications have other ways to enrich

Enrichment Strategies for the Detection of Phosphorylated Peptides Time (min) neutral peptides basic peptides SCX Strong Cation Exchange Chromatography Stationary phase is negatively charged Mobile phase is a buffer that is increasing the pH (if peptide becomes neutral it elutes) Neutral peptides elute earlier: XXpSxxxxxR/K Positive peptides elute late: XXXXHXXXXR/K

Several Strategies are often combined

Multiplexed Mouse Study 10mg of protein for each of the 27 samples

Multiplexed Mouse Study 49 x 3 hr LC-MS analysis (>147 hours of instrument time)

Is the protein or the phosphorylation changed?

What if the enrichment worked, but you still don’t ‘see’ much?

MS/MS is not informative m/z

MS/MS is not informative Peptide with two possible modification sites

MS/MS is not informative Peptide with two possible modification sites MS/MS spectrum m/z

MS/MS is not informative Peptide with two possible modification sites Matching MS/MS spectrum m/z

MS/MS is not informative Peptide with two possible modification sites Matching MS/MS spectrum m/z

MS/MS is not informative Peptide with two possible modification sites Matching MS/MS spectrum m/z

Change the way the peptide get’s dissociated in the mass spectrometer What if enough of the modified peptide is present, but still no ID is possible? Change the way the peptide get’s dissociated in the mass spectrometer

Tandem MS - Dissociation Techniques CAD: Collision Activated Dissociation (b, y ions) increase of internal energy through collisions

Tandem MS - Dissociation Techniques CAD: Collision Activated Dissociation (b, y ions) increase of internal energy through collisions ETD: Electron Transfer Dissociation (c, z ions) bombardment of peptides with electrons (radical driven fragmentation)

Tandem MS - Dissociation Techniques CAD: Collision Activated Dissociation (b, y ions) selective fragmentation ETD: Electron Transfer Dissociation (c, z ions) more random fragmentation

Detecting PTMs In theory quite simple, only the mass of the amino acid changes In practice difficult for several reasons Peptide is too short or too long to be detected Not enough sequence coverage to assign the site of modification Modification is present only in a small percentage Peptide is never selected for MS/MS Several amino acids in the peptide could be modified Modification might interfere with fragmentation Not enough sequence coverage to identify the peptide

Searching for PTMs

CAD versus ETD x x Low charge Modified from Joshua Coon, Analytical Chemistry, 81, 3208-3215 (2009)

Doubly charged peptides

CAD versus ETD x Low charge Labile modifications Modified from Joshua Coon, Analytical Chemistry, 81, 3208-3215 (2009)

Phosphopeptides

O-Sulfonated Peptides

CAD versus ETD x x Low charge Labile modifications Intact proteins Modified from Joshua Coon, Analytical Chemistry, 81, 3208-3215 (2009)

Intact Histone Protein CAD ETD

Intact Histone Protein CAD M M P Ac Ac Ac P M P Ac M Ac M M M P P M M H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50 ETD

Large highly charged peptides 1:1000 +6 415.7 m/z 11 12 13 14 Time (min) 415 m/z