Workshop: SILAC and Alternative Labeling Strategies in Quantitative Proteomics

Workshop: SILAC and Alternative Labeling Strategies in Quantitative Proteomics

SILAC and Stable Isotope Dimethyl-Labeling Approaches in Quantitative Proteomics Ho-Tak Lau, Hyong-Won Suh, Shao-En Ong UW Pharmacology shaoen@uw.edu March 30, 2015

Outline SILAC and Stable Isotope Dimethyl-Labeling, two methods for stable isotope-based quantitative proteomics Developing a StageTip Stable Isotope Dimethyl-Labeling protocol Comparing the two labeling methods Application: Dasatinib kinase inhibitor pulldown Application: Quantitative labeling of gel-separated samples 3

Stable isotopes to label proteomes Ong, SE and Mann, M Nat Protocols 2007 4

SILAC Stable Isotope Labeling by Amino acids in Cell culture Ong SE et. al. Mol Cell Proteomics (2002) 5

Two- & Three-state SILAC Triple encoding SILAC Light Heavy Experiment Ong SE et. al. Mol Cell Proteomics (2002) Blagoev B., Ong SE et. al. Nat Biotech 6 2004

Stable Isotope Dimethyl-Labeling Two-state labeling Hsu, JL et. al. Anal Chem 2003 Three-state labeling Boersma, P et. al. Nat Protocols 2009 7

Features of these quantitative approaches SILAC Incorporating isotope labels in growth medium - Requires living cells for labeling Low cost + Allows measurement of synthesis and turnover in pulse labeling experiments + Samples can be combined early in the experimental design, at whole cells for organelle purification + PTMs do not affect labeling efficiency Stable Isotope Dimethyl-Labeling Chemical labeling of primary amines in peptides + Essentially complete labeling in one pot reaction Low cost + Not metabolic incorporation, can label tissue sample post-digestion - Samples have to be combined after separate enzymatic digest and post-labeling - Modified N-terminal peptides that are not terminated in Lys are not labeled and cannot be quantified. 8

Why compare SILAC and Dimethyl- Labeling? 1. Wanted to use chemical labeling, often looking at pairwise or three-state comparisons 2. Wanted an inexpensive chemical labeling alternative to commercial isobaric tags like itraq and TMT 3. Wanted to label large amounts of peptide (e.g. phosphoproteomics projects ~10 mg/condition) 4. Wanted to develop a robust chemical labeling protocol for use with gel-separated samples 9

Comparing SILAC, Dimethyl Labeling, TMT Precursor MS quantification (SILAC, Dimethyl-Labeling) Reporter ion MS2 quantification (TMT) Result Result Result Result Benchmarking stable isotope labeling based quantitative proteomics Altelaar et. al. J. Proteomics 2013 10

Comparing SILAC, Dimethyl Labeling, TMT Benchmarking stable isotope labeling based quantitative proteomics Altelaar et. al. J. Proteomics 2013 11

Comparing SILAC and mtraq Comparison of SILAC and mtraq Quantification for Phosphoproteomics on a Quadrupole Orbitrap Mass Spectrometer Oppermann et. al. JPR 2013 12

Comparing SILAC and mtraq Comparison of SILAC and mtraq Quantification for Phosphoproteomics on a Quadrupole Orbitrap Mass Spectrometer Oppermann et. al. JPR 2013 13

Comparing methods in single LC-MS runs One sample Lau, HT, Suh, HW et. al. JPR 2014 +28 Da 14

Analytical setup Thermo Dionex RSLCNano UPLC and Autosampler Thermo Orbitrap Elite Self-pulled 360 µm OD x 75 µm ID column with ~10 µm tip Self-packed 10-15 cm long, 3 µm C18-AQ Reprosil (Dr. Maisch) Source with column heater @ 55ºC ( Priska Von Haller PhD, UWPR) 200 nl/min flow, 90 min 3-35% acetonitrile ( 0.1% acetic acid) Varying sample complexity (Unfractionated, and affinity pulldowns) MaxQuant ver.1.3.0.5, searching the same data files with separate sets of search parameters (either SILAC or dimethyl-labeling), then combining result files in R for analysis 15

C18 StageTips peptide desalting Rappsilber J, Mann M, Ishihama Y Nature Protocols 2007 16

StageTip dimethyl-labeling Boersma et. al. 2009 described several dimethyl labeling protocols, including online column labeling, in-solution labeling, and C18 offline SPE cartridge labeling We wanted to miniaturize the labeling reaction to allow quantitative labeling of small amounts of peptide (1 to 15 µg peptide) C18 StageTips already used for desalting of peptide samples prior to LCMS Is offline dimethyl-labeling protocol compatible with StageTips? 17

StageTip dimethyl-labeling protocol works! StageTip dimethyl-labeling results in higher peptide intensities than in-solution labeling (14% higher) Longer, more hydrophobic peptides in StageTip labeling protocol 18

Dynamic range of SILAC & Dimethyl- Labeling Both precursor MS methods show same ratio compression Unfractionated whole cell lysates in 90 min LCMS runs 19

Precision and repeatability Overall precision, normalized peptide ratios Repeatability of protein ratios Precision: Intra-day variation (4 sets); Repeatability: Variation in replicates on different days (3 days) 20

Loss of hydrophilic peptides in Dimethyl-labeling? Unique peptides IDs Peptides unique in SILAC sample are more hydrophilic ~5% lower intensity in Dimethyllabeled vs. SILAC peptides ~23% fewer peptide IDs in Dimethyllabeled samples vs. SILAC 21

Identifying REAL binders in pulldowns Ong SE et. al. PNAS 2009 22

Application to dasatinib pulldowns Coverage of known dasatinib targets Label swap replicates for each set 23

Scatter plots of dasatinib pulldown 24

Both methods identify known targets 25

SILAC (but not dimethyl labeling) identifies false positives in label-swap experiments 26

The end of gel bands Sample A: 10973 detected isotopic clusters Sample B: 11737 detected isotopic clusters 27

Can you reliably compare these samples? Sample A Sample B One gel slice, one LC run Sample A: 2349 identified peptides 402 proteins Sample B: 2453 identified peptides 425 proteins 28

Yes! With StageTip dimethyl-labeling 29

Conclusions Precision (intra-day replicates) and Repeatability (inter-day, biological replicates) are similar in SILAC and stable isotope dimethyl-labeling SILAC samples combined before trypsin digestion provide the highest level of reproducibility Label-swap SILAC experiments provide additional specificity to detect lysate-specific differences in pulldown experiments Developed a robust StageTip Stable Isotope Dimethyl- Labeling protocol for gel separated proteins 30

Acknowledgments ONG LAB Ho-Tak Lau Hyong-Won Danny Suh Martin Golkowski Emily Myers Funding NCI NIAMS NIBIB http://www.quantbiology.org 31

Roundtable Discussion Stable Isotope Labeling Approaches in Proteomics: An Open Forum Panelists: Chris Colangelo, Yale Thomas Neubert, NYU Langone Shao-En Ong, UW Bret Phinney, UC Davis Brian Searle, Proteome Sciences