DAH3.1 Mass Spectrometry Kathryn Lilley Cambridge Centre for Proteomics Department of Biochemistry...
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Transcript of DAH3.1 Mass Spectrometry Kathryn Lilley Cambridge Centre for Proteomics Department of Biochemistry...
DAH3.1 Mass Spectrometry
Kathryn Lilley
Cambridge Centre for Proteomics
Department of Biochemistry
University of Cambridge
www.bio.cam.ac.uk/proteomics/
Part III Systems Biology
Definition of the Proteome
The analysis of the entire PROTEin complement expressed by a genOME.
Wasinger et al Electrophoresis 16 (1995)
Could be:Cellular extractSecreted fluidTissueWhole organism
Why bother studying it????
CCaammbbrriiddggee CCeennttrree ffoorr PPrrootteeoommiiccss
A Proteomicist’s Tools
• Mass spectrometry• Protein and peptide separation methods• Databases and software
• Validation tools – Western blotting– GFP tagging and confocal microscopy
Instruments for mass analysis
Mass Spectrometers measure m/z of gaseous ion
Mass spectrometers comprise:
A sourcewhich is responsible for ionising the sample, e.g. electrospray, laser desorption
An analyserwhich separates and carries the ions to the detectore.g. Quadrupole, Ion-trap (mass range 2000-4000 m/z)TOF (time of flight) (mass range 0-200,000+ m/z)
A detectore.g. electron multiplier
Outline
• Proteomics workflows
• Protein identification
• Post translational modification
Protein analysis on pure proteins/complexes
Mass of protein
Modification statuscan be difficult to deconvolute with many isoforms
Higher order structuresability to spray whole complexes and look at components and
stoichiometries
Low through put methods
Usually carried out on pure proteinsor complexes
Already know what the protein is
Protein analysis on complex mixtures
For more complex samples you cannot purify each one and then analyse it.
Methods need to be applied where proteins can be analysed simultaneously
Proteins can be separated then analysed or converted to peptides which are then analysed
The peptides act as surrogates for the protein
Types of protein analysis
Proteins present PPI SCL Function
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
Arrays
Abundance
Quantitative Mass Spec
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
Arrays
Isoform status
Mass Spectrometry
Western blotting
Functional arrays
Y2H
Tagging + Mass Spectrometry
Western blotting
Structural studies
Protein Arrays
Biophysical assays (e.g.ITC,
AUC)
Mass Spectrometry
GFP tagging
Immuno- histochemistry
Enzyme assay
Functional arrays
Enzyme assay
Genetic approaches
Which proteins are present
Trypsin
Peptides
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
1D gel 2D gel Solution Digest
Workflow 1
• MALDI/MS
• Peptide mass fingerprinting
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
ExciseDigest Apply to MALDI ToF
Matrix Assisted Laser Desorption Ionisation (MALDI)
-cyano-4-hydroxycinnamic acid
Linear Detector
Sample target
N2 Laser
Ion Beam
Matrix Suppression
Lens
Reflectron Detector
Reflectron Assembly
Gas Cell
The chemical matrix absorbs energy from the laser pulse which is transferred to the proteinThe sample ions are then accelerated towards the detectorPrincipally produces M+H+ ions (sometimes M+2H+ )
MALDI Tof MS
Trypsin
K
RR
K
1457.351765.331975.722055/782589.31
Matrix assisted desorption time of flight mass spectrometry
Mass list
Database search of virtual trypsin digested translated genome
Identification !!!!
Peptides
Peptide Mass Fingerprinting
Score = 110
Limitations
Only works well for purified proteins
Require well annotated genome
Strengths
QuickCheap
Workflow 2
• HPLC peptide separation
• Electrospray ionisation
• LC MS/MS
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
Digest
MS CID MS
Chromatography separations
High Performance Liquid Chromatography (HPLC)
Strong cation exchange (SCX)Separation based on net charge of peptide
Weak anion exchange (WAX)Separation based on net charge of peptide
Reverse phase (RP)Separation based on hydrophobicity
Hydrophobic interaction chromatography (HILIC)Separation based on hydrophobicity
Bind peptides
Elute with gradient
e.g. acetonitrile for reverse phaseIncreasing salt for SCX
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
LC-MS/MSMolecular ion (precursor) is accelerated into collision cell where it collides with an inert gasSome of the kinetic energy is converted to internal (vibrational) energyPeptide cleavage takes place largely at the peptide bond nearest a mobile proton
Net result is:
Detect positively charged fragments which contain either the original N-terminus or C-terminus of the peptide
Tandem Mass Spectrometry LC-MS/MS:
Data Dependent Acquisition in MS
Q CID ToF
Precursor ionselection basedon intensity
Precursors scannedout of first quad.
Collision induced dissociation
All fragment ions analysed
Typical output
• List of peptide masses
– Precursor mass (parent ion mass)
• Fragment ion masses
– y-ions– b-ions
Protein identification
• Search engines
MASCOT - http://www.matrixscience.com
SEQUEST - fields.scripps.edu/sequest/
X ! Tandem -www.thegpm.org/tandem/index.html
Phenyx- www.genebio.com/products/phenyx/
Score = 960
MUDPIT
• Data dependent acquisition means that only the most intense ions at any given time are taken for MS/MS
• To improve coverage, peptide simplification is required
MUDPIT
Multi dimensional proteinIdentification technology
Washburn et al (2001)Nat. Biotech 19:242
Strengths and weaknesses
• Can be used with very complex mixtures of proteins
• If the genome is not sequenced then sequence returned may show similarity or identity to related organisms
• De novo sequencing
• More time consuming• Equipment more expensive
Can you be sure?Validation??
• GFPUsing molecular biology techniques fuse gene encoding a
fluorescent protein to your protein of interest.
• Western blotting
Proteins from gel blotted onto PVDF membrane
Primary antibody – raised against your protein of interest
Secondary antibody – raised against the first antibody constant regions
Enzyme, fluorescent tag
Quantitative Western Blotting on a System-wide Scale
Quantitative western blotting of 75% of yeast proteome
Ghaemmaghami et al, 2003
A massive amount of work
Not transferable to many organisms
GFP tagging of yeast proteome
Huh et al, 2003
GFP tagged proteins
75% of the yeast proteome classified to 22 distinct location
Systems wide immuno-
histochemistry
Barbe et al, 2008
Antibodies to 488 proteins applied to 3 different human cell lines and images stored and publically accessible
Blue = DAPI staining of nucleus
Types of protein analysis
Proteins present PPI SCL Function
Mass Spectrometry
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
Arrays
Abundance
Quantitative Mass Spec
Western blotting
GFP tagging
Immuno- histochemistry
Enzyme assay
Arrays
Isoform status
Mass Spectrometry
Western blotting
Functional arrays
Y2H
Tagging + Mass Spectrometry
Western blotting
Structural studies
Protein Arrays
Biophysical assays (e.g.ITC,
AUC)
Mass Spectrometry
GFP tagging
Immuno- histochemistry
Enzyme assay
Functional arrays
Enzyme assay
Genetic approaches
Protein Isoform Analysis
Proteins may be:
– Covalently modified– Truncated– Dimerised
Isoform status
Mass Spectrometry
Western blotting
Functional arrays
Post Translational Modifications
100s of different PTMs
Most commonly characterised
– Phosphorylation xxx– Acetylation/Methylation x– Ubiquitination x– Sumoylation xx– Glycosylation– S-nitrosylation xxxx– …………………….
Isoform status
Mass Spectrometry
Western blotting
Functional arrays
Phosphorylation
Phosphorylation is a very important PTM
Signalling pathwaysProtein conformational changes
Serine, threonine and tyrosine are the most frequently phosphorylated residues
Phosphorylation
Most popular approaches
• 32P incorporation to track peptides and quantify recovery
• Isolate / enrich phosphopeptides by metal-chelation chromatography
• Use triple-quad and hybrid-Tof instruments to look for neutral mass loss
• Prediction algorithms
Problems with Phosphoproteomics
• Phospho groups are highly dynamic
• Phospho tyrosine is very rare
• Phosphopeptides ionise poorly, they tend to be very acidic
• The phosphate group tends to fall off pSer and pThr during MS/MS
Phospho-protein and -peptide enrichment
• Phospho-tyrosine
– good antibodies
• Phospho-serine and phospho-threonine
– Metal chelate chromatography– Ion exchange chromatography
AgaroseBead
Fe3+
NTA
OH
OPO
O
COOH
NH2
Immobilized metal affinity chromatography Titanium (IMAC) Dioxide
Enrichment methods for phosphopeptides
Ferric or Gallium columns most usually employed
Mass Spectrometry of Phosphopeptides
• Standard methods
• Neutral loss
• ETD
Precursor ion Loss of PO3- group Intense fragment ion peak at m/z = 747.94 (2+) m = 98 Da, z = 2+ m/z = 698.94 (747.94 – 49)
m/z = 49
RLSIELTNSLLRP P
RLSIELTNSLLR
Neutral Loss
Electron Transfer Dissociation (ETD).
Gentler fragmentation than CID Preserves post-translational modifications, such as
phosphorylation Produces c and z ions Better sequence coverage than CID
[M + 3H]2+•
[M + 3H]3+ + A- [M + 3H]2+• + A
Electron Transfer Dissociation (ETD)
[C+2H]1+ + [Z+H]1+•
C Z
R
R
>1 eV Electron “Thermal”
e-
-
+
FluorantheneRadical Anion (Good Electron Donor/ETD Reagent)
CID: Prominent loss of phosphate
Parent ion = 571.22
538.25 = loss of phosphoric acid
200 300 500 700 800 1100 1300 1500 1600 1800 1900 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100538.25
559.02
742.61
Cambridge_A1 T:
400 600 900 1000 1200 1400 1700
Cambridge_A1T:Cambridge_A1
65
70
75
80
85
90
95
100
Cambridge_A1
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
(M+3H-H3PO4)3+
KKSSLSSNVGSTVKPPTKLSSNVGSTVKPPTK
ETD:ETD: KKSSLSSNVGSTVKPPTKLSSNVGSTVKPPTK
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
856.18
1711.71
571.03
791.141694.83
1581.70957.40600.31
754.38
1479.611112.54870.421157.58714.42554.32233.32 1058.49
653.44513.29 1399.721286.90
Methylation/Acetylation
Pang et al (2010) Identification of arginine- and lysine-methylation in the proteome of Saccharomyces cerevisiae and its functional implicationsBMC Genomics 2010, 11:92
Choudhary, et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science 325, 834-840.
More straightforward, but will be issues with digestion rates if trypsin is used
Can enrich, antibody affinity capture to acetyl-lysine
Ubiquitin
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
Kirkpatrick D.S., Denison C., and Gygi S.P., Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol, 2005. 7(8): p. 750-7
Ubiquitin is a small highly conserved eucaryote protein, it attaches to other protein via lysine residues by ubiqutin ligases, often marking proteins for degradation by the proteasome system
………….X-X-X-K-X-X……………….
..R-L-R-G-G-
UBIQUITIN
SUBSTRATE PROTEIN
Tryptic digestion
R G D E L Q K G A F LI
GG
SUMO
Small Ubiquitin-like Modifier
3 (4) versions
Many functions including stability, nuclear-cytosolic transport, and transcriptional regulation
Sadly there is no well placed tryptic site or site for any other common protease near the point at which it attaches to its modification target
MSMS spectra are thus a mess, as two sets of –b and –y ions will be produced per with SUMO modification
Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Pedrioli PG, et al. Nat Methods. 2006 3(7):533-9.
Galisson et al (2011) Mol. Cell Prot. A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells
Engineered SUMO is Hek293 cells to have strategically located tryptic site and (His)6 for purification
Bruderer et al EMBO Rep. 2011 Feb;12(2):142-8..Purification and identification of endogenous polySUMO conjugates.
E3 ligase inactive RNF4 fragment binds polySUMO
Glycosylation
Common, up to 50% of human proteins are glycosylated
Need to fond site of attached, using N-linked (Asn) or O-linked (Ser)
Also need to determine structure of glycosyl group
Very complex, highly combinatorial
The most challenging PTM for high throughput proteomics
Enrichment possible with lectin affinity chromatography
Typical schema used in large scale glycoproteome analysis
Sialylation
Dephosphorylation using a phosphatase
Palmisano et al (2010) Nat. Protocols 5
Sialyl groups are sometimes found as end caps of glycan chains.
They are extremely important in recognition between molecules. Sialyl lewis x, for instance, is important in ABO blood antigen determination and correct functioning of the immune response
Sialylation status has also been implicated in metastasis
Truncations
• Easiest way look at peptide coverage
• N-terminal peptide analysis– Edman degradation
COFRADICCombined fractional diagonal
chromatographyAcetylate total proteins with acetic anhydride. All amino
groups acetylatedDigest with trypsin
Carry out liquid chromatography (usually reverse phase) and collect peptides in fractions
Modify all new N-termini generated with trypsin with TNBS (this makes the peptide very hydrophobic).
Rerun all fractions using same LC conditions as before. Peptide which eluted in the same place are the original N-terminus, those that move are internal peptides.
AcAc
Ac
******Gevaert et al (2003) Nat. Biotech 21:566
This week
• Tuesday:– 1pm Seminar by Matthias Mann– 2.15pm Q and A session with Prof. Mann and
lecture for me
• Wednesday:– 9am – Practical Class– 4pm Lecture from me