Electron transfer dissociation (ETD)

Transcription

1 Electron transfer dissociation (ETD)

2 Electron transfer dissociation (ETD) Originally developed for FT-ICR MS as ECD ( C for capture) Now often used on other trapping instruments, even Q-TOFs Trick: Bringing together analyte cations + reagent anions for some time Ion types generated: c and z ions PTMs remain intact (Phosphate, N-glycans, O-glycans...) Works good with very large peptides /proteins top down proteomics

3 Intens. x104 Electron transfer dissociation (ETD) Glycosylation of fusion tag found! Myc and His LISEEDLNSAVDHHHHHH with 4, 6 and 7 Mannoses measured, all showed same glycosylation site between myc and His-tag LISEEDLNSAVDHHHHHH [M+4H] 4+ +MS2(ETD 777.0), min #( ) ' ' ' ' ' ' m/z 2+ '

4 A few more words on identification Example: ID of Prothrombin using GPM found peptides unfindable peptides (too small) missing sequence Sequence coverage in %

5 A few more words on identification Example Prothrombin use of MS/MS spectra

6 A few more words on identification Example Prothrombin etc. etc. log(e)... error probability, the smaller the better; from to ca (Score in Mascot, from about 40 to ca. 2000) --for above example protein log(e) = -850 How to define limit? False discovery rate. Probability value at which the number of identifications in a decoy database (e.g. reverted database) does not exceed a preset percentage (e.g. 1%) PS: ppm mass accuracy obtained by lock mass - calibrant constantly in spray

7 Strategies in proteomics Systems biology- understand cellpathways, network, and complex interacting (includes Genomics, Proteomics, Metabolomics..) Biological processes-characterize protein complexes, cellular machines, organelles, proteins induced by stimuli (e.g. chemokines) Biomarkers - proteins / peptides in urine, serum or tissues associated with a certain disease - for diagnosis and monitoring of therapy Drug targets discover biological processes involved in disease mechanism, which are targets for (rational) drug design (antigonists, inhibitors, antibodies etc.)

8 Strategies in proteomics Identification of proteins

9 Strategies in proteomics Identification of proteins 1.) From gel spots 2D-gels of 1D-gels of total proteins of cells / tissue prefractionated subset (solubility, pi etc.) mostly for immunoprecipitates Advantages: quantitation on gel, pre-identification by size and pi reduced complexity of spots to be analyzed by MS Disadvantages: blind at the extremes of size and pi problems with difficult to solubilize membrane proteins

10 Strategies in proteomics

11 Strategies in proteomics Identification of proteins 2) From total protein mixtures / tissues shotgun proteomics protease digestion performed on whole sample Advantages: includes insoluble proteins (multispan-proteins*), very large or very basic etc. proteins Disadvantages: extremely complex mixture of peptides mix of high and low abundance species Solution: (one of a few) two-dimensional LC before ESI-MS 1. - CIEX with salt pulses 2. - RP of CIEX fractions multi-dimensional protein identification = mudpit * special tricks for digestion of transmembrane region

12 Strategies in proteomics Functional protein complexes Immunoprecipitation Protein A Yeast two hybrid system on the DNA level Blue native PA-electrophoresis (BN-PAGE) for membran-proteins and protein-complexes mild detergent solubilizes + CB charges protein

13 Membrane proteins Membrane proteins in normal buffer solutions not soluble enrichment / fractionation Many membrane proteins can be solubilized with mild detergents e.g. Triton X-100, deoxycholate, octyl-ß-d-glucoside, u.v.a. but for many others these are inefficient. Radical solution: SDS, not suitable for later MS analysis. More general: SDS for dissolving maximum of proteins - removal by aceton + methanol / chloroform precipitation -alternative: FASP... filter aided sample preparation

14 Membrane proteins Blue Native polyacrylamide gel electrophoresis, BN-PAGE Protein-complexes solubilized with detergent (digitonin, dodecyl-maltoside) and Coomassie-blue G-250 CB confers charge to complexes, but does not denature them in contrast to SDS. BN-PAGE may be used as first dimension before SDS-PAGE

15 Comparing samples by 2D-PAGE Run normal 2D-PAGE, one for each sample Stain with silver, or with Sypro Ruby (more even staining of various proteins, better linearity of quantitation; less than 10 ng of protein; also Sypro Orange etc.; requires fluorimetric scanner) Compare the gels by eye or scan and compare with soft-ware Plus: Cheapest method Con: 2D-gels never identical, overlay problematic

16 Comparing samples by 2D-PAGE Example: liver cells (hepatocytes) control and drug treated from a work of Christopher Gerner, Wien 48 pi kda 20

17 Comparing samples by 2D-PAGE 2-D Fluorescence Difference Gel Electrophoresis (DIGE) 2 or even 3 samples can be compared proteins are reacted with a fluorescent dye (either Cy3, Cy5, or Cy6) mixed and separated by 2D-PAGE

18 Comparing samples by 2D-PAGE 2-D Fluorescence Difference Gel Electrophoresis Plus: Samples run on same gel: no gel comparison guess work Sample pool can be used as quantitation standard: allows rather small relative differences in expression level to be measured (lod: ±10 %) Con: Expensive (only from GE healthcare) Only for 2D-PAGE accessible proteins

19 Cy3 Comparing samples by 2D-PAGE Cy5 T-plastin Dual ImageCy3/Cy5 Glutaminase C in breast cancer vs. control

20 Comparing samples by MS or MS/MS Dimethyl-labeling One pot using stable isotopes ITRAQ (or TMT) SILAC Sample by sample label free quantitation (shotgun or targeted)

21 Dimethyl-labeling Tryptic digest of sample 1 Tryptic digest of sample 2 maybe sample 3

22 Relative quantitation of protein expression by stable isotope labeling by amino acids in cell culture (SILAC) 12 C-Arg Arg-0 light Mix MS 13 C 6 -Arg Arg-6 heavy Evaluate peak ratios for various peptides Identify peptides / proteins with significant difference

23 Relative quantitation of protein expression by ITRAQ Tryptic digest of sample 1 Tryptic digest of sample 2 -NH 2 reaction with reagent containing only H -NH 2 reaction with reagent containing only 8 D Mix MS + MS/MS Evaluate peak ratios for various peptides Identify peptides / proteins with significant difference ITRAQ is performed on mixtures of proteins with very good HPLC

24 Principle of ITRAQ or TMT

25

26 Quantitative proteomics by shotgun principle from Clemens Gruber

27 Quantitative proteomics by shotgun principle ---- Discovery proteomics Problem: - only the most abundant proteins are selected for MS/MS - many MS/MS spectra cannot be interpretated results are not reproducible information on a peptide that was not selected is not in data file Solutions: 1.) increase speed and sensitivity of instrument 2.) extend gradient time (up to 4 h with one-dimensional LC) 3.) enhance chromatographic separation (less overlap of peaks) using sub-2 µm LC (minimizes problem of hybrid spectra) 4.) pre-fractionate protein mixture e.g. by cation-exchange LC or RP-HPLC at different ph, e.g. ph 9(shielded silica or polymeric RP-phase) General problem when performing MS/MS on selected precursor: hybrid spectra

28 Other solution: Targeted proteomics" E.g.in clinical diagnosis. protein-specific peptides for the biomarker, e.g. prostate specific antigen, are defined. MRMs and elution times are known AQUA peptides with 13 C isotopes are synthetized (* Absolute QUAntiation) and added to sample Only relevant MRMs acquired high dwell time, high sensitivty Usually done on triple-quad instruments

29 Quantitative proteomics: Third way: MS E and SWATH MS E (Waters) -no precursor selection -switching between MS and MS/MS on full mass range - alignment of precursor and fragment mass by software MS E SWATH (AB Sciex) - no precursor selection - switching between MS and MS/MS on large mass segments - MRM atlas rather then de novo interpretation of spectra prerequisite: very fast instrument

30 New trick: Ion mobility Remember: MS in vacuum, size and shape of ion do not matter Not so in Ion-mobility MS (various technical realizations) ions move against gas molecules Applications: Separation of isomers prior to MS Separation of folding states of a protein

31 Strategies in proteomics Functional protein complexes Immunoprecipitation Protein A Yeast two hybrid system on the DNA level Blue native PA-electrophoresis (BN-PAGE) for membran-proteins and protein-complexes mild detergent solubilizes + CB charges protein

32 Membrane proteins Membrane proteins in normal buffer solutions not soluble enrichment / fractionation Many membrane proteins can be solubilized with mild detergents e.g. Triton X-100, deoxycholate, octyl-ß-d-glucoside or digitonin but for many others these are inefficient. Radical solution: SDS, not suitable for later MS analysis. More general: SDS for dissolving maximum of proteins - removal by aceton + methanol / chloroform precipitation -alternative: FASP... filter aided sample preparation

33 Membrane proteins Blue Native polyacrylamide gel electrophoresis, BN-PAGE Protein-complexes solubilized with detergent (digitonin, dodecyl-maltoside) and Coomassie-blue G-250 CB confers charge to complexes, but does not denature them in contrast to SDS. BN-PAGE may be used as first dimension before SDS-PAGE

34 The end of the proteomics part

35 Glycoprotein - Analysis N-GlcNAc N-glycans (Man, GlcNAc, Gal, Fuc, Neu5Ac, GalNAc) O-GalNAc: mucin-type O-glycans (EPO, IgA...) O-fucose: can be involved in signal-reception O-mannose: e.g. in brain O-glucose: Xyl-Xyl-Glc, e.g. on some clotting factors O-xylose: in linkage region of proteoglycans O-GlcNAc: on cytosolic / nuclear proteins IgM with glycan (C. Oostenbrinck)

36 Congenital disorders of glycosylation Mutations in enzymes of protein- or lipidglycosylation can led to severe, often letal defects. A few can be cured by dietary uptake of the relevant sugar (CDG type 1b with mannose) The extreme significance of complex glycans is also demonstrated by the fate of k.o.-mice. WT mouse GnT-I k.o. mouse

37 Polysialic acid in brain development Neuronal cell adhesion molecules (N-CAMs) mediate connections between neuronal cells in nerves and brain. In embryos (= developing brain) the cells must be able to change binding partners = plasticity This requires partial inhibition of protein-protein interaction by sugar polymers (poly-sialic acid).,.

38 Leukocyte rolling Inflammation starts with invasion of leukocytes to the affected tissue. This process is initiated by the presentation of certain sugar epitopes on neighboring endothelial cells. 38

39 Sugar binding proteins snap shot II Adhesion of bacteria to urogenital as well as intestinal epithelia Receptors on host tissues are glycolipids and/or glycoproteins

40 N-glycan trimming in the ER ,,.!

41 The case of erythropoietin. 41

42 alfa-galactose, - α-β-β-.,!

43 How to treat the glyco protein? protein glycan holistic ESI-MS of whole glycoprotein

44 ( )

45 reductionist I --with proteases ESI-MS of whole glycopeptides Denaturation S-Alkylation Trypsin digestion Essentially a proteomic workflow...yet, the Scope of the Data- Mining is different! Site-directed glycosylation analysis (any type of glycan)

46 glycan reductionist II -- with peptide:n-glycosidase

47 reductionist II -- with peptide:n-glycosidase glycan Release of N-glycans with PNGase Normal-phase HPLC (HILIC) CZE HPAEC HPLC MALDI carbon-lc-esi-ms

48 Analysis by hydrophilic liquid interaction chromatography (HILIC) Synonyme: Normal-phase; sizing HPLC start: ca. 80 % acetonitrile gradient to ca. 50 % acetonitrile R O HO OH OH NH O H N CONH 2 Example shows IgG glycans on a 1.8 µm UPLC column (Agilent) (R. Figl, March 2017) sugars labeled with a fluorescent dye Fluorescence (m mv) GnGnF AGnF GnAF AAF GnNaF ANaF Time (min) GlcNAc Man Gal Fuc Neu5Ac

49 Analysis by porous graphitic carbon chromatography (PGC-LC) coupled to electrospray ionization MS PGC: Sort of RP-matrix capable of binding glycans (at least disaccharides) high shape selectivity for glycans isomer separation Diantennary N-glycans on PGC EIC bovine fibrin Cow SIM intensity Time (min) Pabst et al.2007, Anal. Chem. 79 Mass + Retention time = Structure...

50 Analysis by porous graphitic carbon chromatography (PGC-LC) coupled to electrospray ionization MS PGC-LC-ESI-MS works with virtually each and all kinds of glycans (neutral + sialylated) Diantennary structures with alfa-gal unlike MALDI-TOF-MS SIM 895.9

51 Part: Altmann: Title: Analysis of Posttranslational protein modifications I Date: Viennese speciality: LC-ESI-MS on graphitic carbon SIM Diantennary structures with alfa-gal Time (min) Basic Course II Biosynthesis, Post-translational Modification and Trafficking of recombinant Proteins

52 Type of ion greatly influences result: Analysis of glycans by MSMS - H -adducts in positive mode: re-arrangements!!! - Na-adducts in positive mode: no re-arrangements - H-adducts in negative mode, no artefacts, ring-cleavages (good!) charge state also influences result -very helpful: permethylated glycans R-OH R-O-CH 3 Me Me

53