Mikkel Nissum, Ph.D. - proteomic.org · Proteomics 9 Mikkel Nissum Separation Fractionation...

Mikkel NissumMikkel Nissum, , PhPh.D..D.

2Proteomics Mikkel Nissum

Proteomics

…. the complexity and dynamics of a cell :…. the complexity and dynamics of a cell :

• Approx. 30.000 human genes correspondto more than 200.000 protein species

• Magnitude of abundance of a proteinspecies between 1 and 106

• Protein expression is highly dynamic

• Dynamic protein modifications(phosphorylation, deamidation, etc.)


HeartLiver

LungSkin

KidneyBrain

.... .... many many ProteomesProteomes

One Genome.....One Genome.....

Proteomics


Reducing Complexity Through Pre-Fractionation

Isolation of cell organelles„ Subcellular Proteomics“

e.g. Free Flow Electrophoresis (ZE)

Fractionation of the protein mixture„Cellular Proteomics“

e.g. Free Flow Electrophoresis (IEF)


Proteomics – A Possible Solution

Tecan-Munich GmbH, Kirchheim

Page 18

Fractionation Fractionation inclincl. . sample preparationsample preparation

SeparationSeparation1.1. DimDim. IEF. IEF

GelGel--CastingCastingSDSSDS--PAGEPAGE

SeparationSeparation2.2. DimDim. SDS. SDS--PAGEPAGE

StainingStainingPAGEPAGESpotSpot--PickingPicking

In gelIn gel--digestdigest

MALDIMALDI--MSMS

A possible ProTeam Configuration


Proteomics – Reduction of Complexity

Proteome

Free Flow ElectrophoresisPro Team FFE

2D Polyacrylamide ElectrophoresisPro Team 2D-PAGE

Protein Processing/DigestPro Team Digest

Mass Spectrometry MALDI - ESI

DATA


Free Flow Electrophoresis

Eno1Ssb1

Tdh1

Cdc10

HIS1

HIS2

CDC37

RNA1

FAS1

YER049w

ARP3

End3

ARP7

AdhTdh3


Free Flow Electrophoresis

counterflow

stabilization medium

separationmedium

sample inlet

ANODE

CATHODE

fractioncollector

counterflow

stabilization medium

separationmedium

sample inlet

ANODE

ANODE

CATHODE

CATHODE

fractioncollector


Separation

FractionationFractionation SeparationSeparation QuantificationQuantification ProcessingProcessing AnalyticAnalytic

1. Dimension1. Dimension The ProTeam IEF

(Iso-Electric Focussing)

Solution:• Highly reproducible • Complete automation• Powerful protein separation range

(zoom gels)• Reagents/Kits: IEF-Strips, Buffers,

etc.


Separation

FractionationFractionation SeparationSeparation QuantificationQuantification ProcessingProcessing AnalyticAnalytic

The Pro Team PAGESolution:• Tightly controlled gel casting• Reproducible parallel

electrophoresis• Effective and sensitive gel staining• Complete automation of

electrophoretic separation• Reagents/Kits: PA, Buffers, Staining,

etc.

2. Dimension2. Dimension


The Process of Protein Identification

2D-Gel Spot Picking

Digestion MALDI-TOF Analysis


The Process of Protein Identification

MALDI-TOF Spectra – Database Search


C2, Phos B

B2, Phos B

F1, Phos B

H2, Ovo

D2, Ovo

H1, Ovo

F2, Transf

C3, Transf

D3, TransfB1, Ovo

E2, Ovo

G2, Perox

B4, Perox

G3, Lac Dehy

A2, Lac Dehy G1, Lac Dehy

C1, CAA3, CA

E1, CA

A4, Beta-Lac

H3, Beta-Lac

A1, Not ident.

B3, Not ident.

D1, Not ident.F3, Not ident.E3, Not ident.

A1, D1, B3: Same spectrum but no identification (SwissProt, All org.)E3, F3: Same spectrum but no identification (SwissProt, All org.)

Identifications of Proteins


In-Gel Digestion - Principles

Enzymatic Digestion:TrypsinChymotrypsinArg-CAsp-NLys-CPepsin.....

Peptide Mass Fingerprintor

MS/MS


The Digestion Process

• As many protocols as users!!• Basic steps are always the same

Destaining Spotting onMALDI-targetPurificationExtractionDigestionShrinking

Process Overview

Load Gel Plugs

Reduction and Alkylation


Gel pieces in MTP

Reduction and Alkylation50 µL 50 mM ammonium bicarbonate/30% acetonitrile

5 µL 10 mM TCEP in 50 mM ammonium bicarbonate/30% acetonitrileMix and incubate 30 min at RT

5 µL 40 mM iodoacetamide in 50 mM ammonium bicarbonate/30% acetonitrileMix and incubate 30 min at RT in the dark

Remove liquid (Vacuum)Wash with 50 mM ammonium bicarbonate/30% acetonitrile

Remove liquid (Vacuum)

START

Destaining50 µL 50 mM ammonium bicarbonate/30% acetonitrile

Incubate 5 min at 37 °CRemove liquid (Vacuum)

Repeat steps

Dehydration80 µL 80% acetonitrileIncubate 10 min at RT

Remove liquid (Vacuum, 60 °C Incubator)

Digestion4 µL of trypsin 12.5 ng/µL

Wait for 10 min for gel to swellRemove excess trypsin (Vacuum)

4 µL 5mM Tris-HCl, pH 8.0Incubate 2 hours at 37 °C

Acidification4 µL 1% TFA

Incubate 15 min at RT

Extraction of PeptidesZipTips......

Purification of Peptides10 µL 0.1% TFA

Elution of Peptides on MALDI-TOF Target(400 µm or 600 µm AnchorChip, Bruker)

1-2 µL 0.09 g/L CHCA in 90% acetonitrile/0.1% TFA

END

The Digestion Process - Details


Pro Team Digest:Automation of in-gel digestion

Pro Team Digest


Pro Team Digest

Spotting onMALDI-target


Pro Team Advanced Digest


Pro Team Advanced Digest:TecPro96TM – TecPrep96TM Plate Combination

• TecPro96TM - Processing plate for the initial handling of gel piecesV-shaped 96 well MTP containing at the bottom of each well a capillary with a diameter of 75 µm. The capillaries allow liquid to be removed from the wells using vacuum. The small diameter of the capillaries prevent evaporation.

• TecPrep96TM - SPE plate for concentrating and clean-up of peptidesV-shaped 96 well MTP similar to the TecPro96TM but capillaries contain C18 modified ChromolithTM. ChromolithTM is a monolithic solid phase made of a highly porous silica rod.


Pro Team Advanced Digest – Vacuum Manifold

Design of Wash Adapter prevents contamination.

The Repositioner makes it possible to spot on all 384 positions on the Bruker AnchorChipTM Target.


Performance of the TecPrep96TM Plate

• A peptide mixture was provided in avolume of 4 µl. A minimum volumeof 1% TFA was added for acidification. Peptides were then bound to the adsorbing material. Subsequently, the peptides were washed and eluted directly onto aBruker 400 µm AnchorChip target.

• Peptides are identified in the low attomol range.

• The mentioned quantities are per peptide.

500 fmol

10 fmol

1 fmol

500 amol

100 amol

100 fmol


Processing of BSA from a Coomassie Stained 1D-Gel

• Different amounts of BSA were loaded onto a SDS-PAGE gel and run under standard conditions. The gel was stained with coomassie blue.

• Parts of the BSA containing bands were excised andprocessed with the TecPro96TM

– TecPrep96TM plate com-bination on the Pro Team Advanced DigestTM platform from Tecan.


Processing of BSA from a Coomassie Stained 1D-Gel

2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol

# PeptidesSequence CoverageBSA

2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


2342 %20 fmol

2239 %30 fmol

2851 %40 fmol

4271 %100 fmol

4067 %200 fmol

4467 %400 fmol

2138 %4 pmol


Spectrum of 20 fmol BSA


Processing of Transferrin from a Silver Stained 1D-Gel

50 fmol TransferrinSequence Coverage: 50 %


Processing of Gel Pieces with theTecPro96TM – TecPrep96TM Plate Combination

• Gel pieces were excised from a coomassie stained yeast 2D-gel pH 4-7 and processed using the TecPro96TM – TecPrep96TM

plate combination.

• Intensive as well as weak spots were processed.

• Some examples are shown on the next pages.


Processed 2D-Gel Pieces

Processed spots are marked on the 2D gel (only part of the gel shown)

A

BC

D

EF

H

I

G


PMF of 2D-Gel Spot (Spot A)

Proteasome component Y7


PMF of 2D-Gel Spot (Spot B)

Phosphoribosylamidoimidazole-succinocarboxamide synthase


PMF of 2D-Gel Spot (Spot C)

Spermidine synthase


PMF of 2D-Gel Spot (Spot D)

SEC14 cytosolic factor


PMF of 2D-Gel Spot (Spot E)

Twinfilin A


PMF of 2D-Gel Spot (Spot F)

Mixture of 2 proteins Protein phosphatases PP1regulatory subunit SDS22

ADP-ribosylation factorGTPase-activating protein GCS1


PMF of 2D-Gel Spot (Spot G)

Glucokinase


PMF of 2D-Gel Spot (Spot H)

Probable ATP-dependent RNA helicase SUB2


PMF of 2D-Gel Spot (Spot I)

Transcriptional modulator WTM1


Summary of Identified Proteins

0.1431 %Proteasome component Y7A

0.2359 %Phosphoribosylamidoimidazole-succinocarboxamide

synthase

B

0.3659 %Spermidine synthaseC

0.3071 %SEC14 cytosolic factorD

0.1446 %Twinfilin AE

I

H

G

F

Spot

Transcriptional modulator WTM1

Probable ATP-dependent RNA helicase SUB2

Glucokinase

Protein phosphatases PP1 regulatory subunit SDS22

+ ADP-ribosylation factor GTPase-activating protein

GCS1 - MIXTURE

Protein

0.2547%

0.3758 %

0.1655 %

0.18 and 0.1555 % and 68 %

Codon biasSeq. coverage


In-Gel Digestion of Rat Liver Proteins froma SYPROTM Ruby Stained 2D-Gel

• A 2D-gel with a pH gradient from 4-7 was prepared from rat liver. The gel was stained with

SYPROTM Ruby. Spots with strong, medium and weak intensities were picked with the

GelPixTM Spotcutter from Genetix. In-gel digestion of the proteins were performed on the

Pro Team Advanced DigestTM using the TecPro96TM – TecPrep96TM plate combination.

Samples were eluted directly onto a Bruker 600 µm AnchorChipTM target. MALDI-TOF

analysis including acquisition of PMF and MS/MS spectra was done using a Bruker

ULTRAFLEXTM mass spectrometer. Proteins were identified applying the Mascot search

engine on the SwissProt database.

• Examples of identified proteins are shown. In some cases MS/MS spectra were acquired

to increase confidence of the search result (MS/MS Peaks). Proteins from faint SYPROTM

Ruby stained spots were identified. This illustrates the sensitivity of the system. Intensive

spots were processed reliably indicating sufficient capacity of the TecPrep96TM plate.


Identified Proteins from the SYPROTM Ruby Stained 2D-Gel

153011301193

91111481131

8891269963

1481 986

1584 1072

1227 920

pH 4 pH 7 MW


Identified Proteins from the SYPROTM Ruby Stained 2D-Gel

Spot ID Protein % Sequence coverage MS/MS Peaks1269 3-hydroxyanthranilate 3,4-dioxygenase 44 0963 Prohibitin 75 01193 S-adenosylmethionine synthetase 31 01530 Protein disulfide isomerase A3 46 01130 Aldehyde dehydrogenase, mitochondrial 43 01131 F-actin capping protein alpha-2 subunit 58 01481 Ornithine carbamoyltransferase, mitochondrial 34 11148 Guanine nucleotide-binding protein 25 11584 Proteasome subunit beta type 4 37 2

1072 Thioredoxin-dependent peroxide reductase, mitochondrial

32 2

889 Biliverdin reductase A 18 1

920 LMW phosphotyrosine protein phosphatase ACP1/ACP2 59 2

911 Annexin V 57 21227 Serum amyloid P-component 15 1986 3-alpha-hydroxysteroid dehydrogenase 42 1

Score1522757815915813020598145

170

53

204

208121103

Spot ID Protein % Sequence coverage MS/MS Peaks1269 3-hydroxyanthranilate 3,4-dioxygenase 44 0963 Prohibitin 75 01193 S-adenosylmethionine synthetase 31 01530 Protein disulfide isomerase A3 46 01130 Aldehyde dehydrogenase, mitochondrial 43 01131 F-actin capping protein alpha-2 subunit 58 01481 Ornithine carbamoyltransferase, mitochondrial 34 11148 Guanine nucleotide-binding protein 25 11584 Proteasome subunit beta type 4 37 2

1072 Thioredoxin-dependent peroxide reductase, mitochondrial

32 2

889 Biliverdin reductase A 18 1

920 LMW phosphotyrosine protein phosphatase ACP1/ACP2 59 2

911 Annexin V 57 21227 Serum amyloid P-component 15 1986 3-alpha-hydroxysteroid dehydrogenase 42 1

Score1522757815915813020598145

170

53

204

208121103

Mikkel Nissum, Ph.D. - proteomic.org · Proteomics 9 Mikkel Nissum Separation Fractionation...

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