Quan%ta%ve proteomics. Maarten Altelaar, 2014

Transcription

1 Quan%ta%ve proteomics Maarten Altelaar, 2014

2 Proteomics Altelaar et al. Nat Rev Gen 14, 2013, 35-48

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4 Quan%ta%ve proteomics

5 Quan%ta%ve proteomics Control Diseased, s%mulated, Knock down, etc. How quan%ta%ve is mass spectrometry?

6 Quan1fica1on requires internal standards or controlled condi1ons All pep%des and proteins will experience DIFFERENT levels of loss during a proteomic experiment In most cases the level of sample loss changes in each proteomic experiment Stable Isotope labelling was introduced to overcome the above obstacles. Isotopomeric pep%des behave almost iden%cal throughout the experiment EXCEPT for the mass spectrometric analysis.

7 Basics : Natural Elemental Isotopes In nature, most elements are present in more than one isotopic form: e.g. Carbon is present in two stable forms 12 C and 13 C and one unstable 14 C which is radioac%ve and used for carbon da%ng. Mass spectrometry can dis%nguish these isotopes since they have different mass Element Isotope Relative Abundance Isotope Relative Abundance Carbon 12 C C 1.11 Hydrogen 1 H H.016 Nitrogen 14 N N.38 Isotope Relative Abundance Oxygen 16 O O O.20 Sulfur 32 S S S 4.40 Chlorine 35 Cl Cl 32.5 Bromine 79 Br Br 98.0

8 Basics : Natural Elemental Isotopes Appearance of spectra: CH 4 CH4: C1 H4 p(gss, s/p:40) Chrg 1R: C 20 H 40 C20H42: C20 H42 p(gss, s/p:40) Chrg 1R: Res CH 3 Cl CClH3: C1 Cl1 H3 p(gss, s/p:40) Chrg 1R: Res CH 3 35 Cl Relative Abundance Relative Abundance Relative Abundance CH 3 37 Cl CH C CH CH 3 Cl C C 2 H m/z m/z m/z

9 ADTQLLLLR Isotopically labeled Unlabeled Ra%o = 2 Mass difference

10 Where and How to Introduce Stable Isotopes Biological incorpora1on Chemical incorpora1on (SILAC/Metabolic)(ICAT) (itraq, MassTag) Absolute quan%fica%on instead of rela%ve quan%fica%on can be performed if the level of the internal standard is known.

11 SILAC:Stable Isotope Labeled Amino Acids in Culture Incorpora%on of heavy isotopes is performed by uptake of amino acids by the cells from the media Require the cell type to be auxotrophic for that amino acid. Typical amino acid(s) chosen for SILAC are: Leucine; one of the most common amino acids present in pep%des Lysine and Arginine; all pep%des will have at least one labeled amino acid if trypsin is used. e.g. 12 C 13 C 13 C 13 C H 2 N 13 C 13 C OH O 13 C 6 leucine 12 C 12 C 12 C 12 C H 2 N 12 C 12 C OH O regular leucine

12 SILAC:Stable Isotope Labeled Amino Acids in Culture

13 SILAC:Stable Isotope Labeled Amino Acids in Culture Light Heavy Certain cell types can convert arginine into proline. In those systems pep%des that contain prolines will have mul%ple peaks for the heavy pep%de.

14 Label swap A B + Log ra%o reversed B A unchanged - + Log ra%o forward - Specific

15 Pijnappel et al. Nature (7442):

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17 A B

18 Metabolic Labeling of Arabidopsis thaliana 1: Metabolic labeling with 15 N (KNO 3, <5% NH 3 ) 14 N 14 N 15 N Requires cell types that do not extract amino acids from the media Typical media use: 15N Potassium Nitrate for Arabidopsis thaliana 15N Ammonium Sulfate for Sacchromyces Cerevisiae

19 Metabolic Labeling of C. elegans

20 Metabolic Labeling of Mammals

21 SILAC Labeling of Mammals

22 Quan1ta1on through Chemical Deriva1sa1on There are many, many,...many ways and all have advantages and disadvantages.

23 ICAT Chemical labeling : the iodoacetyl group reacts selec%vely with Cys residues of proteins. ICAT Reagents: Heavy reagent: d8- ICAT (X=deuterium) Light reagent: d0- ICAT (X=hydrogen) O N S N O N X X O X X O X X O X X N O I Bio1n tag Linker (heavy or light) Thiol specific reac1ve group Reagent has been modified to use 13C instead of 2H or D due to deuterium containing pep%des elu%ng earlier in reverse phase chromatography than their normal analogues.

24 Dimethyla1on formaldehyde X peptide NH 2 + O X peptide N X X + H O H NaCNBH3 H peptide N + X H O + X H X X peptide N H X X H + O X X NaCNBH3 peptide X N X H X H X Dimethylates lysine residues and N- termini. Current experimental condi%ons: (100 mm TEAB or sodium acetate) Semi- tolerant towards dirty samples. (Some types of sample can ini%ate polymerisa%on.) Allows labelling of whole digests in 10mins.

25 Relative Abundance RT: Normal Dimethyla%on Dimethyla1on Time (min) Likle change in hydrophobicity of methylated pep%des. Not necessary to clean up sample aler labelling. Likle change in MSMS spectra NL: 3.68E6 Base Peak F: ITMS + c NSI Full ms [ ] MS _SM3017_ 14 NL: 1.91E6 Base Peak F: ITMS + c NSI Full ms [ ] MS _sm3017_ 15

26 On- line dimethyla1on

27 Peak picking Peak integra%on Mass accuracy Resolu%on SoZware Relative Abundance z= z=2 z= z= z=2 z= z=2 z= z= z= m/z RT: Relative Abundance Time (min) MS MS MSMS Relative Abundance Relative Abundance z= z= z=2 z= z= z= z= z= z= m/z z= z= z= z= z=2 z= m/z

28 OR4_110703_EDG_11EDEG007_IP_3d # 7688 RT: AV: 1 NL: 1.56E4 F: FTMS + p NSI Full ms [ ] Relative Abundance m/z OR4_110703_EDG_11EDEG007_IP_3d # 7699 RT: AV: 1 NL: 2.02E4 F: FTMS + p NSI Full ms [ ] Relative Abundance m/z OR4_110703_EDG_11EDEG007_IP_3d # 7693 RT: AV: 1 NL: 1.84E4 F: FTMS + p NSI Full ms [ ] Relative Abundance m/z OR4_110703_EDG_11EDEG007_IP_3d # 7703 RT: AV: 1 NL: 1.49E4 F: FTMS + p NSI Full ms [ ] Relative Abundance m/z OR4_110703_EDG_11EDEG007_IP_3d # 7709 RT: AV: 1 NL: 1.50E4 F: FTMS + p NSI Full ms [ ] Relative Abundance m/z

29 Large scale quan1ta1ve proteomics analysis; stem cells Munoz et al. Molecular Systems Biology 2011

30 Dimethyl Technical Reproducibility Tech Rep 2: Progenitor/iPS Tech Rep 2: ESC/iPS Tech Rep 1: Progenitor/iPS Tech Rep 1: ESC/iPS