Proteomic based approaches in developmental biology
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Proteomic Based Approaches In Developmental Biology. 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.

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Proteomic Based Approaches In Developmental Biology

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Proteomic Based Approaches In Developmental Biology


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.


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.


1394.49

8000

7000

6000

1099.34

1252.42

5000

Abundance (Intensity)

861.24

1742.46

1584.48

4000

989.30

750.24

2846.95

3000

2522.78

1787.43

2000

1000

500

1000

1500

2000

2500

3000

(m/z)

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).


Mass Spectrometry Data

From Andrew Link, Vanderbilt University


Basic Mass Spectrometer

From “Mass Spectrometry in Biotechnology by Gary Siuzdak


Fundamental Parts of a Mass Spectrometer

Ion source

Mass analyzer

Detector

1. TOF

2. Ion trap

3. Quadrupole

4. Orbitrap

5. Magnetic sector

6. FTICR

1. MALDI

2. ESI


Ionization Techniques

From “Mass Spectrometry in Biotechnology by Gary Siuzdak


Properties of Different

Mass Analyzers


Ionization

Steen and Mann, 2004


m/z Not Enough

b-ion

y-ion

Verification of all proteins must be done by second phase MS to obtain sequence data of at least 3 peptides per protein

O O O O

C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH

=

=

=

=

CH3

CH3

CH2-OH

CH2-CH-(CH3)2

Peptide: SLAA


m/z Not Enough

Verification of all proteins done by second phase MS to obtain sequence data of at least 3 peptides per protein.

y-ion

y-ion

y-ion

O O O O

C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH

=

=

=

=

CH3

CH3

CH2-OH

CH2-CH-(CH3)2

Peptide: SLAA

e.g. y-ion series=A, AA, LAA, SLAA


m/z Not Enough

b-ion

b-ion

b-ion

b-ion

Verification of all proteins done by second phase MS to obtain sequence data of at least 3 peptides per protein.

O O O O

C-NH-CH-C-NH-CH-C-NH-CH-C-NH-CH

=

=

=

=

CH3

CH3

CH2-OH

CH2-CH-(CH3)2

Peptide: SLAA

y-ion series= A, AA, LAA, SLAA

b-ion series=S, SL, SLA, SLAA


TBX20 MS/MS Raw Data

~115D + H20 (16D) =

133D; i.e. Aspartic Acid

~115D


How do you what proteins you have?


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).


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.


How do you get the protein sequence?


How do you know where your protein sequence came from?

How do you know where your protein 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

MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSWRVISSIEQKTMADGNEKKLEKVKAYREKIEKELETVCNDVLSLLDKFLIKNCNDFQYESKVFYLKMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEQMQPTHPIRLGLALNFSVFYYEIQNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDE

EAGEGN


Analysis with xProteo of Tbx20-GFP (MALDI)Source: 293 cells (Human Kidney Cell Line)

False Positive

Junk


Further Look Into TBX20 Data


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.


Overview of Approach


Work Flow

Can go from isolating tissue to trypsin in 1 day

(a very long day)

Incubation with Ab, 1hr at 4C. Takes another ~8hrs to wash, elute and concentrate (20ul)


How Much Tissue is Enough?


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


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


Initial Lysis Buffers Routinely Used

Worked

With

TBX20


Affinity Purification


Which Epitopes or Tags

  • Ideal: High affinity, high specificity antibody against endogenous protein.

  • 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)


Avi-tag Tagged Tbx20

Avi-tag epitope knocked into Tbx20 locus


Biotinylation by BirA

Biotin

Biotin

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


AP Tagged Tbx20

Construct knocked into Tbx5 containing mouse for Tg


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.


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


Best for separating whole cell lysate- looking at in tact proteins!

You will lose Membrane proteins, histones, highly acidic proteins


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.

  • 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 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.


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 (O16/O18 labeling)

  • Isobaric (equal mass) peptide tags

    • iTRAQ &TMT


SILAC Labeling Reagents

  • 13C- and 12C-Lysine(heavy – light = 6 D)

  • 13C- and 12C-Arginine(heavy – light = 6 D)

C12->C13 adds oneneutronC13 is chemically indistinguishable


SILAC

(98%)

Prostate cancer cell line PC3

PC3M

(low metastatic potential)

PC3M-LN4

(high metastatic potential)

Everley et al., MCP 2004


Labeling Efficiency of both isotopes

Ratio = 0.98

Doublet is separated


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


iTRAQ: Isotope Coding


8-plex isobaric tagging reagents: iTRAQ


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


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