mikkel nissum, ph.d. - proteomic.org · proteomics 9 mikkel nissum separation fractionation...
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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.)
3Proteomics Mikkel Nissum
HeartLiver
LungSkin
KidneyBrain
.... .... many many ProteomesProteomes
One Genome.....One Genome.....
Proteomics
4Proteomics Mikkel Nissum
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)
5Proteomics Mikkel Nissum
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
6Proteomics Mikkel Nissum
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
7Proteomics Mikkel Nissum
Free Flow Electrophoresis
Eno1Ssb1
Tdh1
Cdc10
HIS1
HIS2
CDC37
RNA1
FAS1
YER049w
ARP3
End3
ARP7
AdhTdh3
8Proteomics Mikkel Nissum
Free Flow Electrophoresis
counterflow
stabilization medium
separationmedium
sample inlet
ANODE
CATHODE
fractioncollector
counterflow
stabilization medium
separationmedium
sample inlet
ANODE
ANODE
CATHODE
CATHODE
fractioncollector
9Proteomics Mikkel Nissum
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.
10Proteomics Mikkel Nissum
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
11Proteomics Mikkel Nissum
The Process of Protein Identification
2D-Gel Spot Picking
Digestion MALDI-TOF Analysis
12Proteomics Mikkel Nissum
The Process of Protein Identification
MALDI-TOF Spectra – Database Search
13Proteomics Mikkel Nissum
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
14Proteomics Mikkel Nissum
In-Gel Digestion - Principles
Enzymatic Digestion:TrypsinChymotrypsinArg-CAsp-NLys-CPepsin.....
Peptide Mass Fingerprintor
MS/MS
15Proteomics Mikkel Nissum
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
16Proteomics Mikkel Nissum
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
17Proteomics Mikkel Nissum
Pro Team Digest:Automation of in-gel digestion
Pro Team Digest
18Proteomics Mikkel Nissum
Pro Team Digest
Spotting onMALDI-target
19Proteomics Mikkel Nissum
Pro Team Advanced Digest
20Proteomics Mikkel Nissum
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.
21Proteomics Mikkel Nissum
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.
22Proteomics Mikkel Nissum
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
23Proteomics Mikkel Nissum
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.
24Proteomics Mikkel Nissum
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
# PeptidesSequence CoverageBSA
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
# PeptidesSequence CoverageBSA
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
# PeptidesSequence CoverageBSA
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
# PeptidesSequence CoverageBSA
Spectrum of 20 fmol BSA
25Proteomics Mikkel Nissum
Processing of Transferrin from a Silver Stained 1D-Gel
50 fmol TransferrinSequence Coverage: 50 %
26Proteomics Mikkel Nissum
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.
27Proteomics Mikkel Nissum
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
28Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot A)
Proteasome component Y7
29Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot B)
Phosphoribosylamidoimidazole-succinocarboxamide synthase
30Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot C)
Spermidine synthase
31Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot D)
SEC14 cytosolic factor
32Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot E)
Twinfilin A
33Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot F)
Mixture of 2 proteins Protein phosphatases PP1regulatory subunit SDS22
ADP-ribosylation factorGTPase-activating protein GCS1
34Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot G)
Glucokinase
35Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot H)
Probable ATP-dependent RNA helicase SUB2
36Proteomics Mikkel Nissum
PMF of 2D-Gel Spot (Spot I)
Transcriptional modulator WTM1
37Proteomics Mikkel Nissum
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
38Proteomics Mikkel Nissum
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.
39Proteomics Mikkel Nissum
Identified Proteins from the SYPROTM Ruby Stained 2D-Gel
153011301193
91111481131
8891269963
1481 986
1584 1072
1227 920
pH 4 pH 7 MW
40Proteomics Mikkel Nissum
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
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