# Presentation: 526631

## Functional Genomics UCB-LBNL

**Functional Genomics UCB-LBNL**

- Jay Keasling, Core Leader
- Vincent Martin, Scientist
- Aindrila Mukhopadhyay, Post-doc
- Lianhong Sun, Post-doc
- Alyssa Redding, Graduate student

## FGC UCB-LBNL current aims

- 	Develop the methods to measure the metabolite profile (metabolome) of *D. vulgaris *during the response of various environmental stresses.

- 	Use proteomics (ICAT) methods to measure differential protein expression profiles of *D. vulgaris* during the response of various environmental stresses.

- 	Generate mutants of the various genes whose gene products are though or found to be responsible for a particular stress response.

## Acknowledgements

- Aindrila Mukhopadhyay, Post-doc, Proteomics and Genetics
- Lianhong Sun, Post-doc, Metabolomics
- Alyssa Redding, Graduate student, Proteomics and Genetics

## Metabolite profiling (Metabolomics)

- Metabolite extractions from microbial cells *(E.coli and D. vulgaris)*
- Approach to and difficulties in measuring metabolites from *D. vulgaris*
- Analysis of CoenzymeA’s and Nucleotides

_**Definition of a metabolome**_
- Total complement of metabolites in a cell at any given growth condition

## Metabolite extraction from microbial cells

**Stable**
**Intermediates**

**Cells**

**Supernatant**

**Secreted**
**Metabolites**

**Cytoplasm**

**Insoluble **
**Fraction**

**Metabolite Amounts**
**And Labeling Patterns**

**Amino Acid**
**Labeling Pattern**

**Total Macromolecule**
**Content (DNA, RNA, Lipid,**
**Storage Polymers**

**Centrifugation**

**HPLC-MS**

**Lysis**

**HPLC-MS**
**CE-MS**

**Derivitization**
**GC-MS**

**GC-MS**
**HPLC-MS**

**Quench Metabolism**

## Metabolites extraction from microbial cells

- A good extraction method will:
- Extract the largest number of metabolites
- Be non-selective
- Be non-destructive
- Be very rapid (turnover of ATP in cell is in msec)

-   Commonly used methods:
- - Hot ethanol or methanol (70-90°C)
- - Cold methanol (-40°C)
- - Perchloric and trichloroacetic acids
- - Alkali (KOH)
- - Methanol/chloroform

## Metabolite extractions from E. coli

- Metabolite extractions from *E. coli*

- TCA in D2O

-  Quickly centrifuge at 4° C

-  Transfer TCA layer to 2 ml
-   centrifuge tube and neutralize

- Analysis

_**Advantages:**_
-  Very fast quenching of metabolism
-  Concentrates metabolites in samples
-  Separate cells and supernatant

## Metabolite extractions from D. vulgaris

- TCA in D2O

- Silicone oil

- Iron sulphide (FeS) precipitate
-  accumulates at the surface of silicone oil

-  No or little metabolites present in TCA

**Therefore resorted to simple TCA extractions**

## Analytical instruments for measuring metabolites from D. vulgaris

**GC-MS (EI or CI)**
- High sensitivity, resolution and good selectivity
- Requires compound volatility (derivatization)
**HPLC-UV-vis**
- Low sensitivity and selectivity
**HPLC-MS-MS (ESI)**
- High sensitivity and selectivity
- Problems with buffer compatibility with LC
**CE-MS**
- High sensitivity, resolution and good selectivity

## HPLC separation

- Reverse Phase
- Buffer: NH4OAc
- Organic phase: Acetonitrile
- Column: C-18 (0.3mm150mm)
- Hydrophilic Interaction
- Buffer: NH4OAc
- Organic phase: Acetonitrile
- Amide-80 (2  250 mm)+ flow splitter
- Ion-pair reverse phase
- Tetrabutyl ammonium salt
- Not compatible with electro-spray ionization
- Ion-Exchange
- Suppressed ionization in electro-spray

## Grouping of metabolites

| Class | Separation Methods |
| --- | --- |
| CoA’s | Reverse-phase HPLC-MS |
| Nucleotides | Hydrophilic interaction HPLC-MS
CE-MS |
| Nucleotide Sugars | Ion-exchange HPLC-MS
Ion-pair Reverse Phase HPLC-MS
CE-MS |
| Neutral Sugars | GC-MS of TMS derivatives |
| Redox Cofactors | Hydrophilic interaction HPLC-MS
CE-MS |
| Glycolytic Pathway Intermediates | Ion-exchange HPLC-MS
CE-MS |
| Organic acids | Ion-exclusion HPLC-MS
CE-MS |
| Amino Acids | GC-MS of derivatives
Ion-exchange HPLC-MS |

##  LIT scan modes

## Mass spectrometry scan modes

**MS scan** → Record all ions

** ****Multiple Reaction Monitor (MRM) scan**** → **Select an ion, fragment   			          and select a fragment ion

_**MS-MS**_
**Product ion scan** → Select an ion, 					  fragment & scan

## Multi-component analysis 

- Pairs of ions

- Multi-component analysis

## MS-MS of CoA’s in MRM mode

|  | Molecular Ion
MS (m/z) | Tail
MS/MS (m/z) |
| --- | --- | --- |
| CoA | 768 | 261 |
| Acetyl-CoA | 810 | 303 |
| Propionyl-CoA | 824 | 317 |
| Malonyl-CoA | 854 | 347 |
| Succinyl-CoA | 867 | 361 |

- Coenzyme A   *m/z=768*

- Tail *m/z=261*

- Head  *m/z= 428*

## Capillary LC-MS-MS of CoA’s in positive MRM mode

- Buffer A: 10 mM NH4OAc, pH=5.0 Buffer B: 50% Acetonitrile in buffer A Column: Cap. RP C-18 (0.3  150 mm)  Detection: MRM positive mode Chromatographic conditions:
- 10 minutes at 1% Buffer B, followed by a linear gradient to 20% buffer B in 20 minutes

- Standard references

*D. vulgaris* TCA extract

## MS-MS of nucleotides in positive MRM mode

|  | MS (m/z) | MS/MS (m/z) |  | MS (m/z) | MS/MS (m/z) |  | MS (m/z) | MS/MS (m/z) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ATP | 508 | 136 | GTP | 524 | 152 | CTP | 484 | 112 |
| ADP | 428 | 136 | GDP | 524 | 152 | CDP | 404 | 112 |
| AMP | 348 | 136 | GMP | 364 | 152 | CMP | 324 | 112 |
| dATP | 492 | 136 | dGTP | 508 | 152 | dCTP | 404 | 112 |
| TTP | 483 | 81 | TDP | 403 | 81 | TMP | 323 | 81 |
| UTP | 485 | 97 | UDP | 405 | 97 | UMP | 325 | 97 |
| dUTP | 469 | 97 |  |  |  |  |  |  |

- ATP  *m/z 508*

- Adenine  *m/z 136*

## Capillary LC-MS-MS of nucleotides in positive MRM mode

- Buffer A: 5 mM NH4OAc, pH=5.0;    Buffer B: AcetonitrileColumn: Amide-80 (2  250 mm)Detection: MRM positive mode Chromatographic conditions: 20% buffer A for 10 minutes followed by a linear gradient to 60% buffer A in 20 minutes.

- Standard references

*D. vulgaris* TCA extract

## Metabolomics future directions

- Optimize metabolites extractions for *D. vulgaris*
- Quantitative analysis of CoA’s and nucleotides
- Dynamic range
- Sensitivity
- Reproducibility
- Further development of methods (HPLC,MS,CE)
- Faster, more sensitive, compatible with each other
- More classes of metabolites
- Glycolysis, amino acids, carbohydrates
- Apply the methods to *D. vulgaris* cell extracts

## Proteomics

- _Applied Biosystems_
- QSTAR- quadropole / time-of-flight (TOF)
- High Mass accuracy
- Better resolution

- _LC Packings_
- Nano-LC (nanoL/min)
- Lower limit of detection: ~ 50 femto moles
- Column multiplexing (IE and RP)
- 1D or 2D separation of peptides

## ICAT Reagents

- Free Cystines
-  alkylated

- Biotin tag used
- for enrichment

- X = D0 Light reagent
- X = D8 Heavy reagent

- 13C (0) Light reagent
- 13C (9) Heavy reagent

- Affinity
- Tag
- (Biotin)

- Cleavable
- Linker

- Isotope coded Tag
- (C10H17N3O3)

- Heavy: 9 X 13C (236 amu)

- Light: 9 X 12C (227 amu)

- Protein reactive Group

- Protein

- Composition of reagent: C43H70N7O12SI

## ICAT Strategy

- 1:1 or 1:2

- Cleave
- Biotin Tag

- 1D or 2D Liquid
- Chromatography

- Cleanup
-       &

## Preliminary results Bacillus subtilis sporulation ICAT experiment

- 1 : 2

- 1 : 1

- +3 charged
-    peptide

- t0  vs t30

## MS-MS data and sequence for peptide

## Proteomics (ICAT) experiments

## What can we expect from Bacillus ICAT experiment?

- What was found: 40 proteins but none were in the above list

## What about Desulfovibrio?

- 20 HSK which contain the conserved H, N D/F and G boxes.

## Still working on sample preparation
Lysis protocol
Protein fractionation

- Lysis protocol
- Protein fractionation

- The future of proteomics (ICAT) experiments

- Increase protein coverage
- Moving from 1D to 2D separation on the nanoLC to increase protein coverage

- First set of data from the O2 stressed experiment

## Developing genetic systems for D. vulgaris

- Higher throughput creation of mutants
- The established way....SacB and conjugation
- Method being tested
- Method being proposed:
- One step integration
- Wanner system

-   Reporter system(s) and regulated
-    promoters in *D. vulgaris*

-   Label mutants with barcode

## The established way....SacB and conjugation

- ori

- Amp

- 5% Sucrose

*sacB*

- Cm

*E.coli *ori

- Cm resistance

*D. vulgaris* chromosome

- Cloning of gene and insertion of the resistance cassette takes too long

- Conjugation and for two-step integration takes too long

- Insertion elements inactivate *sacB* and result in false +ive

_**Not suitable for large scale**_

## Rapid cloning method being tested for gene knockout 

- Method is being tested on three cloned Histidine Sensor Kinases

**One PCR**
**No Ligase**

**One plasmid prep**
**Two PCR**
**No Ligase**

## Proposed one-step integration system

- Essential *Bacillus subtilis* genes
- PNAS 2003 100: 4675-83

- 4100 genes knocked out

- CmR

- oriT

- CmR

- oriT

*D. vulgaris* chromosome

**One cloning step,**
**no ligase**
**Single cross-over (no need for SacB)**
**Reporter for *****D. vulgaris***** promoter**
**Downstream polar effect identified**

## Other fun things on the list

- Test electroporation

- Test reporter systems
- Fluorescent substrate (diffusion into *D. vulgaris*)
- GFP