1. Proteomic Based Approaches In Developmental Biology

2. Why Proteomics? Alterations between cells, tissues, and embryos often are not associated with changes in RNA levels, i.e. you cannot answer everything by RNA-seq; protein stability, protein localization, changes in PTMs, etc. Only approximately 10% of all RNAs with changes of 1.5X or greater between two samples, from yeast to human, lead to a change of 1.5X in protein levels. Establish in vivo interactomes as function of stage and tissue. Define in vivo PTMs as function of stage and tissue.

3. Proteomic Based Approaches Proteomics Generally refers to a survey of all the proteins in a given tissue or cell type. Directed Proteomic The identification of a set of proteins that are in complex with a defined protein of interest. Quantitative Proteomics Characterization of the relative amount of a set of proteins between 2-16 samples.

4. Mass Spectrometry – A Brief Definition Mass Spectrometry is a technique for the production of charged molecular species in vacuo, and their separation by magnetic and/or electric fields based on the mass to charge ratio (m/z).

6. Mass Spectrometry Data From Andrew Link, Vanderbilt University

7. Basic Mass Spectrometer From “Mass Spectrometry in Biotechnology by Gary Siuzdak

8. Fundamental Parts of a Mass Spectrometer Ion sourceMass analyzerDetector 1. MALDI 2. ESI 1. TOF 2. Ion trap 3. Quadrupole 4. Orbitrap 5. Magnetic sector 6. FTICR

9. Ionization Techniques From “Mass Spectrometry in Biotechnology by Gary Siuzdak

10. Mass Separation Mass Accuracy Resolution FWHH Scan Rate m/z Range TOFVery good 10-100 ppm Medium 20,000 Very fast (  s) Very wide Ion trapGood 100-1000 ppm Medium 20,000 Fast (m s) 0 – 4000 Quadrupol e Good 100-1000 ppm Low 2,000 Normal (s) 0 – 4000 FTICRExcellent 0.1-1 ppm High >100,000 Normal (s) Very wide Properties of Different Mass Analyzers

11. Ionization Steen and Mann, 2004

12. m/z Not Enough Verification of all proteins must be done by second phase MS to obtain sequence data of at least 3 peptides per protein O O C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH === CH 2 -OHCH 2 -CH-(CH 3 ) 2 CH 3 Peptide: SLAA = CH 3 y-ionb-ion

13. m/z Not Enough Verification of all proteins done by second phase MS to obtain sequence data of at least 3 peptides per protein. O O C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH === CH 2 -OHCH 2 -CH-(CH 3 ) 2 CH 3 Peptide: SLAA e.g. y-ion series=A, AA, LAA, SLAA = CH 3 y-ion

14. m/z Not Enough Verification of all proteins done by second phase MS to obtain sequence data of at least 3 peptides per protein. O O C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH === CH 2 -OHCH 2 -CH-(CH 3 ) 2 CH 3 Peptide: SLAA y-ion series= A, AA, LAA, SLAA b-ion series=S, SL, SLA, SLAA = CH 3 b-ion

15. TBX20 MS/MS Raw Data ~115D ~115D + H20 (16D) = 133D; i.e. Aspartic Acid

16. How do you what proteins you have?

17. Protein Databases Database must contain what you need and little more: “Additional” proteins crashes specificity and discovery rate, increase shared tryptic peptides. Additional” proteins greatly(!) increase time of analysis. Need all available protein sequences from species of interest derived from trEMBL and SWISSPROT (Everything that is in NCBI, Sanger and EMBL).

18. Where Do Protein Sequences Come From? And How Do They Translate Gene Sequences? All protein sequences in NCBI, etc. come from the UniProtDatabase which is divided into: TrEMBL (automated annotation). Contains more redundancy, includes sequence fragments and isoforms. Especially useful for organisms that are not well annotated. Swiss-Prot sequences (manually annotated). Sufficient for well-annotated organisms, e.g. human, mouse, yeast, E.coli, HSV-1 and Xenopus. This is not derived from the sequencing of proteins.

19. How do you get the protein sequence?

20. How do you know where your protein sequence came from? Protein Entry Headers >SOURCE(sp/tr)|ACCESSION|UNIPROT_ID DESCRIPTION ORGANISM GENE >sp|Q04917|1433F_HUMAN 14-3-3 protein eta OS=Homo sapiens GN=YWHAH PE=1 SV=4 MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSWRVISSIEQKTMADGNEKKLEKVKA YREKIEKELETVCNDVLSLLDKFLIKNCNDFQYESKVFYLKMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEQMQPT HPIRLGLALNFSVFYYEIQNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDE EAGEGN How do you know where your protein came from?

21. Analysis with xProteo of Tbx20-GFP (MALDI) Source: 293 cells (Human Kidney Cell Line) False Positive Junk

22. Further Look Into TBX20 Data

23. Proteomic Based Approaches Proteomics Generally refers to a survey of all the proteins in a given tissue or cell type. Directed Proteomic The identification of a set of proteins that are in complex with a defined protein of interest. Quantitative Proteomics Characterization of the relative amount of a set of proteins between 2-16 samples.

24. Overview of Approach

25. Work Flow Incubation with Ab, 1hr at 4C. Takes another ~8hrs to wash, elute and concentrate (20ul) Can go from isolating tissue to trypsin in 1 day (a very long day)

26. How Much Tissue is Enough?

27. Tissue Isolation Need Large/Unlimited Source of Protein for Initial Optimization e.g. TBX20: 293 Cells transfected with pcDNA 3.1-TBX20 CMV promoter driving epitope tagged Tbx20 Starting Material: N=15-20 X 150mm Dishes After Optimization: Current record from mouse of an endogenous protein 4fM (50 adult brains) Selimi et al. 2009 More likely: 500-1500 embryos Alternative: ES cell differentiation

28. Why so much? Each step needs to be optimized for an individual protein.

29. Initial Lysis Buffers Routinely Used Worked With TBX20

30. Affinity Purification

31. Which Epitopes or Tags 1.Ideal: High affinity, high specificity antibody against endogenous protein. 1.V5, Myc and Flg (1,3, 9X) Poor. Commercial Abs of too low affinity and/or low specificity. (V5 worked with Tbx20 but pulled down some non-specific proteins). 2. Anti-HIS (3, 6, 9X); i.e. Nickel or Cobalt Horrific. Very, very high non-specific interactions even in presence of DNAse. If it does work will probably strip off any interacting proteins. 4. HA (3X). Good. e.g. Bienvenu F. et al. 2010, 5. GFP. Better/Best but not an epitope tag and may interfere with protein function, e.g. Selimi et al. 2009 (worked with Tbx20). 6. AVI-Tag (In Progress)

32. Avi-tag Tagged Tbx20 Avi-tag epitope knocked into Tbx20 locus

33. Biotinylation by BirA Streptavidin Applications of BirA-mediated biotinylation Isolation and purification of small poplulations of cells from living embryos Purification of protein complexes Identification of Targets Genes, i.e.Chrommatin IP Biotin

34. AP Tagged Tbx20 Construct knocked into Tbx5 containing mouse for Tg

35. Proteomic Based Approaches Proteomics Generally refers to a survey of all the proteins in a given tissue or cell type. Directed Proteomic The identification of a set of proteins that are in complex with a defined protein of interest. Quantitative Proteomics Characterization of the relative amount of a set of proteins between 2-16 samples.

36. Overcomes the differential gel running problem. You can use software to quantitate

37. Best for separating whole cell lysate- looking at in tact proteins! You will lose Membrane proteins, histones, highly acidic proteins

38. MS-based Quantitative Methods Precursor: Quantitation based on the relative intensities of extracted ion chromatograms (XICs) for precursors within a single data set. This is a widely used approach, which can be used with any chemistry that creates a precursor mass shift. For example, 18O, AQUA, ICAT, ICPL, Metabolic, SILAC, etc., etc.Precursor:18OAQUAICATICPLMetabolicSILAC Reporter: Quantitation based on the relative intensities of fragment peaks at fixed m/z values within an MS/MS spectrum. For example, iTRAQ and Tandem Mass TagsReporter:iTRAQTandem Mass Tags Replicate: Label free quantitation based on the relative intensities of extracted ion chromatograms (XICs) for precursors in multiple data sets aligned using mass and elution time.Replicate:

39. Stable Isotope Labeling Methods ‘Mass difference’ approaches –Metabolic (Stable Isotope Labeling with Amino acids in Cell culture SILAC)- introduces heavy isotopes into sample –Chemical labels (Isotope Coding with Affinity tags ICAT) –Enzymatic (O 16 /O 18 labeling) Isobaric (equal mass) peptide tags –iTRAQ &TMT

40. SILAC Labeling Reagents 13C- and 12C-Lysine (heavy – light = 6 D) 13C- and 12C-Arginine (heavy – light = 6 D) C12->C13 adds one neutron C13 is chemically indistinguishable

41. SILAC Everley et al., MCP 2004 Prostate cancer cell line PC3 PC3M (low metastatic potential) PC3M-LN4 (high metastatic potential) (98%)

42. Ratio = 0.98 Labeling Efficiency of both isotopes Doublet is separated

43. You need to passage cells 3-4 times in heavy media, measure incorporation

44. iTRAQ: Isotope Coding

45. 8-plex isobaric tagging reagents: iTRAQ

46. Great Free On-line Resource: “The Expanding Role of Mass Spectrometry in Biotechnology by Gary Siuzdak