Part III Systems Biology
DAH3.1 Mass Spectrometry
Kathryn Lilley
Cambridge Centre for Proteomics
Department of Biochemistry
University of Cambridge
k.s.lilley@bioc.cam.ac.uk
www.bio.cam.ac.uk/proteomics/
C
a
m
b
r
i
d
g
e
C
e
n
t
r
e
f
o
r
P
r
o
t
e
o
m
i
c
s
Definition of the Proteome
Why bother studying it????
The analysis of the entire PROTEin
complement expressed by a genOME.
Wasinger et al Electrophoresis 16 (1995)
Could be:
Cellular extract
Secreted fluid
Tissue
Whole organism
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 source
which is responsible for ionising the sample, e.g. electrospray,
laser desorption
An analyser
which separates and carries the ions to the detector
e.g. Quadrupole, Ion-trap (mass range 2000-4000 m/z)
TOF (time of flight) (mass range 0-200,000+ m/z)
A detector
e.g. electron multiplier
Outline
• Proteomics workflows
• Protein identification
• Post translational modification
Protein analysis on pure
proteins/complexes
Mass of protein
Modification status
Low through put methods
Usually carried out on
pure proteins
or complexes
can be difficult to deconvolute with many isoforms
Already know what the
protein is
Higher order structures
ability to spray whole complexes and look at components and
stoichiometries
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
Mass
Spectrometry
Western
blotting
Abundance
Quantitative
Mass Spec
Western
blotting
Isoform status
PPI
SCL
Function
Mass
Spectrometry
Y2H
Mass
Spectrometry
Functional
arrays
Tagging + Mass
Spectrometry
GFP tagging
Enzyme assay
Western
blotting
Immunohistochemistry
Genetic
approaches
Enzyme assay
Western
blotting
GFP tagging
GFP tagging
Functional
arrays
Immunohistochemistry
Immunohistochemistry
Structural
studies
Enzyme assay
Enzyme assay
Protein Arrays
Arrays
Arrays
Biophysical
assays (e.g.ITC,
AUC)
Which proteins are present
Mass
Spectrometry
Western
blotting
GFP tagging
Immunohistochemistry
1D gel
2D gel
Enzyme assay
Trypsin
Peptides
Solution Digest
Workflow 1
Mass
Spectrometry
Western
blotting
• MALDI/MS
GFP tagging
Immunohistochemistry
• Peptide mass fingerprinting
Enzyme assay
Excise
Digest
Apply to MALDI ToF
Matrix Assisted Laser Desorption
Ionisation (MALDI)
a-cyano-4-hydroxycinnamic acid
MALDI Tof MS
Reflectron
Detector
Matrix
Suppression
Lens
Sample target
Gas
Cell
Ion Beam
N2 Laser
Reflectron
Assembly
Linear
Detector
The chemical matrix absorbs energy from the laser pulse which is transferred to the protein
The sample ions are then accelerated towards the detector
Principally produces M+H+ ions (sometimes M+2H+ )
Peptide Mass Fingerprinting
K
R
R
Trypsin
Peptides
K
Matrix assisted desorption time
of flight mass spectrometry
Identification !!!!
Database search of virtual trypsin
digested translated genome
1457.35
1765.33
1975.72
2055/78
2589.31
Mass list
Score = 110
Strengths
Quick
Cheap
Limitations
Only works well for purified proteins
Require well annotated genome
Workflow 2
Mass
Spectrometry
Western
blotting
• HPLC peptide separation
• Electrospray ionisation
GFP tagging
Immunohistochemistry
• LC MS/MS
Enzyme assay
Digest
MS
CID
MS
Chromatography separations
Bind peptides
Mass
Spectrometry
Western
blotting
High Performance Liquid Chromatography (HPLC)
Strong cation exchange (SCX)
Separation based on net charge of peptide
GFP tagging
Immunohistochemistry
Enzyme assay
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
Elute with gradient
e.g. acetonitrile for
reverse phase
Increasing salt for SCX
LC-MS/MS
Molecular ion (precursor) is
accelerated into collision cell where it
collides with an inert gas
Some of the kinetic energy is converted to
internal (vibrational) energy
Peptide 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 Cterminus of the peptide
Tandem Mass Spectrometry LC-MS/MS:
Data Dependent Acquisition in MS
Q
Precursor ion
selection based
on intensity
CID
ToF
Collision
induced
dissociation
Precursors scanned
out of first quad.
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 protein
Identification 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??
• GFP
Using molecular biology techniques fuse gene encoding a
fluorescent protein to your protein of interest.
• Western blotting
Enzyme, fluorescent tag
Secondary antibody – raised against the first antibody
constant regions
Primary antibody – raised against your protein of interest
Proteins from gel blotted onto PVDF membrane
Quantitative Western Blotting on a
System-wide Scale
Quantitative western blotting of
75% of yeast proteome
A massive amount of work
Not transferable to many organisms
Ghaemmaghami et al, 2003
GFP tagging of
yeast proteome
GFP tagged proteins
75% of the yeast proteome classified to 22 distinct location
Huh et al, 2003
Systems wide
immunohistochemistry
Antibodies to 488 proteins applied
to 3 different human cell lines and
images stored and publically
accessible
Blue = DAPI staining of nucleus
Barbe et al, 2008
Types of protein analysis
Proteins present
Mass
Spectrometry
Western
blotting
Abundance
Quantitative
Mass Spec
Western
blotting
Isoform status
PPI
SCL
Function
Mass
Spectrometry
Y2H
Mass
Spectrometry
Functional
arrays
Tagging + Mass
Spectrometry
GFP tagging
Enzyme assay
Western
blotting
Immunohistochemistry
Genetic
approaches
Enzyme assay
Western
blotting
GFP tagging
GFP tagging
Functional
arrays
Immunohistochemistry
Immunohistochemistry
Structural
studies
Enzyme assay
Enzyme assay
Protein Arrays
Arrays
Arrays
Biophysical
assays (e.g.ITC,
AUC)
Isoform status
Mass
Spectrometry
Protein Isoform Analysis
Proteins may be:
Western
blotting
Functional
arrays
– Covalently modified
– Truncated
– Dimerised
Isoform status
Post Translational Modifications
100s of different PTMs
Mass
Spectrometry
Western
blotting
Functional
arrays
Most commonly characterised
–
–
–
–
–
–
–
Phosphorylation
Acetylation/Methylation
Ubiquitination
Sumoylation
Glycosylation
S-nitrosylation
…………………….
xxx
x
x
xx
xxxx
Phosphorylation
Phosphorylation is a very important PTM
Signalling pathways
Protein 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
Enrichment methods for phosphopeptides
Immobilized metal affinity chromatography
(IMAC)
COOH
OH
O
P O
O
Fe3+
NTA
NH2
Agarose
Bead
Ferric or Gallium columns most usually employed
Titanium
Dioxide
Mass Spectrometry of Phosphopeptides
• Standard methods
• Neutral loss
• ETD
Neutral Loss
RLSIELTNSLLR
P
Precursor ion
m/z = 747.94 (2+)
RLSIELTNSLLR
P
Loss of PO3- group
m = 98 Da, z = 2+
m/z = 49
Intense fragment ion peak at
m/z = 698.94 (747.94 – 49)
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
Electron Transfer Dissociation (ETD)
[M + 3H]3+ + A[M + 3H]2+•
[M + 3H]2+• + A
[C+2H]1+ + [Z+H]1+•
C
Z
R
Fluoranthene
Radical Anion
(Good Electron
Donor/ETD Reagent)
>1 eV Electron
“Thermal”
e-
R
+
-
CID: Prominent loss of phosphate
(M+3H-H3PO4)3+
Parent ion = 571.22
538.25
Cambridge_A1
T:
538.25 = loss of phosphoric acid
100
95
90
85
80
75
KSLSSNVGSTVKPPTK
Relative Abundance
70
65
60
55
50
45
40
35
30
25
20
15
559.02
742.61
10
5
0
200
300
400
500
600
700
800
900
1000
1100
m/z
1200
1300
1400
1500
1600
1700
1800
1900
2000
ETD: KSLSSNVGSTVKPPTK
856.18
100
95
90
85
80
75
70
Relative Abundance
65
60
1711.71
571.03
55
50
45
40
35
30
25
791.14
1694.83
20
754.38
15
554.32
513.29
233.32
5
1581.70
1479.61
1112.54
1157.58
1058.49
1286.90 1399.72
957.40
600.31
10
870.42
714.42
653.44
0
200
400
600
800
1000
m/z
1200
1400
1600
1800
2000
Methylation/Acetylation
More straightforward, but will be issues with digestion rates if trypsin is used
Can enrich, antibody affinity capture to acetyl-lysine
Pang et al (2010) Identification of arginine- and lysinemethylation in the proteome of Saccharomyces cerevisiae
and its functional implications
BMC Genomics 2010, 11:92
Choudhary, et al. (2009) Lysine acetylation targets protein
complexes and co-regulates major cellular functions, Science
325, 834-840.
Ubiquitin
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
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
UBIQUITIN
Tryptic digestion
………….X-X-X-K-X-X……………….SUBSTRATE PROTEIN
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
R
G
D
E
L
Q
K
G
G
G A
F
LI
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
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
Dephosphorylation
using a phosphatase
Sialylation status has also been
implicated in metastasis
Palmisano et al (2010)
Nat. Protocols 5
Truncations
• Easiest way look at peptide coverage
• N-terminal peptide analysis
– Edman degradation
COFRADIC
Combined fractional diagonal
chromatography
Acetylate total proteins with acetic anhydride. All amino
groups acetylated
Digest 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.
Ac
Ac
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