SILAC and TMT. IDeA National Resource for Proteomics Workshop for Graduate Students and Post-docs Renny Lan 5/18/2017

Transcription

1 SILAC and TMT IDeA National Resource for Proteomics Workshop for Graduate Students and Post-docs Renny Lan 5/18/2017

2 UHPLC peak chosen at min LC Mass at chosen for MS/MS MS/MS MS

3 This is a good start but there are some caveats for label free proteomics The peak is very jittery The day to day and long-term instrument reproducibility is not very good Experimental reproducibility and digestion efficiency Every peptide has its own ionization efficiency (equal amounts of two distinct peptides could have a signal difference of 100x) Instantaneous matrix effects (suppressing or augmenting ion signal by surrounding backgound)

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5 Stable Isotopes: Nature s Gift to Mass Spectrometrists

6 Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Feed cell growth medium with heavy amino acids Proteins incorporate these amino acids All proteins are slightly heavier but otherwise biochemically equivalent Choice of Lys and Arg typical because of trypsin All sample preparation steps happen in parallel Output is a ratio (relative abundance) of a protein or a peptide Carol A. Rouzer. 2015

7 SILAC Applications Protein interaction profiling PTM expression profiling Bait Absent Bait Present Immunoprecipitation Peptides Enrichment

8 Isobaric Tags: TMT and itraq itraq = isobaric Tags for Relative and Absolute(?) Quantification TMT = Tandem Mass Tag Isobaric = same nominal mass Highest plex level: TMT-10plex

9 TMT Duplex Quantitation TMT Sixplex Quantitation TMT 10plex Quantitation

10 Tandem Mass Tags for multiplexed Proteomics

11 Six reporter ions quantify same peptide from six samples in one spectrum

12 10plex with 75 min run: 10 times faster in machine time and data acquisition 150 hours vs 15 hours in 12 fraction experiment

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14 Procedure schematic for using the Thermo Scientific TMT10plex Label Reagents

15 Sample Preparation (Most Important Step!) There is no single protocol for cleaning up the protein samples! Reproducibility Reduce Sources of Variability: Technical: good hands < bad hands Few steps < many steps Biological: cells < tissue < body fluids yeast < nematode < human Fractionation (reduce protein amount and complexity, enrich for target proteins) Buffer Exchange (remove non-compatible buffer reagents)

16 Buffer Reagents Compatibility Depends on the Proteomic Method Three non-compatible categories Detergents Salts Reagents that compete for label Buffer exchange to make compatible Filtration (FASP protocol) Precipitation Binding Affinity Detecting low abundance proteins requires Clean samples, no interfering reagents

17 Potential interference substances Thiols (for example, DTT and mercaptoethanol) High amounts of detergents and denaturants Alternative Detergent/Denaturant (Concentration Limit at Trypsin Digestion) SDS (0.05%) OG (octyl B-D-glucopyranoside) (0.1%) NP -40 (0.1%) Triton X-100 (0.1%) Tween 20 (0.1%) CHAPS (0.1%) Urea (<1M) Note: When using urea, always use a fresh solution. When reducing a sample containing urea, incubate the tubes at 37 C for 1 hour

18 Avoid using any reagents containing Amine containing lysis buffer: Ammonium acetate Ammonium bicarbonate Ammonium citrate Ammonium tartrate AMPD [2-amino-2-methyl-1,3- propanediol] Aminoguanidine bicarbonate salt AMP [2-amino-2-methyl-1- propanol] Ethanolamine Gly-gly Tris buffers primary amines Alternative Buffer : BES BICINE Boric acid CHES DIPSO EPPS HEPBS HEPES HEPPSO MOBS MOPS Phosphate Buffered PIPES POPSO

19 Filter Aided Sample Preparation (FASP) and TMT Labeling Sample protein amount (2 mg): Cell Pellet: 50 ul (15 cm dish) Tissue: 25 mg minced; Freeze/thaw; Pestle; Cryo Bead Homogenizer Serum: Abundant Protein Depletion Protease /phosphatase inhibitor Lysate Preparation: 100 mm DTT 2% SDS Heat (95 C) 3 min FASP processing: 30K filter unit Denaturation: 8M Urea Alkylation: 0.05M IAA Washing: Urea and AMBIC Overnight Digestion: Trypsin Stop reagent: Formic acid Digestion clean up: Sep-Pak Vac C18 (50 mg) High throughput: manifold Peptide Quant DNA Shredding: Sonication QIAshredder UHPLC Clean up TMT Labeling Orbitrap Fusion Protein Quant (100 μg) Bradford or BCA Qubit 3.0 NGS Combine Samples Ratio Check(MS 3 ) High ph fractionation

20 Ratio check Small amount of each TMT labelled samples was mixed in equal volume Either sum of intensity or median can be used for ratio check Correction is needed before mixing

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22 Randomized More than 10 samples (with replicates) Biological replicate Block 1 Block 2 Each block includes all experimental and control groups Block 1 Block 2 Pooled sample Block 1 Block 2

23 Comparison of Quantification Methods Method Advantages Disadvantages Applications Label-free areaunder the curve No special steps required Cost effective SILAC Reduces technical variability Biology does the wok TMT No special cell growth conditions Can use on primary tissue Higher multiplex Synthetic Peptides Guaranteed observation Better handle on actual quantity in sample Label-free Spectral Counting Poor reproducibility Requires special software to keep track of everything Cells must grow in special media Only two channels Noisier than SILAC Compression Can be costly Can interfere with some enrichment strategies Must know analyte(s) of interest Cost $ and time to make the peptide Nothing special Varies widely based on instrument and acquisition method Biomarker discovery when labeling is too expensive Any cell culturebased study Primary cells/tissuebased studies Targeted proteomics Not recommended

24 Isotopic tags: constant mas shift Summary Isobaric (same nominal mass) chemical tags: reporter + normalizer eluted and ionized together Both SILAC and TMT labeling enable relative quantitation and identification simultaneously with multiple conditions Quantify thousands of proteins from 10 samples in a single run TMT can be used with a variety of samples including cells, tissues, and biological fluids SILAC can be used only in cell culture studies but introduces less noise Avoid detergent, salt and reagent interfere label FASP protocol can be used with both SILAC and TMT labeling QC: protein quantification, peptide quantification and ratio check Add pooled sample for analysis more than one batch of TMT