Download proteomics_intro

Introduction to Proteomics
CSC8309 - Gene Expression and
Proteomics
Simon Cockell
Bioinformatics Support Unit
Feb 2008
Outline
• Introduction
– Why proteomics?
• Sample Collection
• Separation Techniques
– Gels
– Columns
• Mass Spectrometry
– Ionisation
– Mass Analysis
– Protein Identification
The proteome
• Organisms have
one genome
• But multiple
proteomes
• Proteomics is the
study of the full
complement of
proteins at a
given time
Why proteomics?
• Microarrays are easier, and more
established
– So why use proteomics at all?
• It is proteins, not genes or mRNA, that
are the functional agents of the genome
• Transcriptome information is only
loosely related to protein levels
– Abundant transcripts might be poorly
translated, or quickly degraded
Basic principles
• 3 steps to most proteomics
experiments
– Preparation of a complex protein
mixture
– Separation of protein mixture
– Charaterisation of proteins within
mixture
Sample Collection
• Controlled conditions
• Low-salt (for later Mass Spec)
• Prevention of:
– Contamination
– Degredation
• Consider difficult to purify proteins
– e.g. membrane-bound
Separation Techniques
2D Gel Electrophoresis
Separation Techniques
2D-GE - Isoelectric Focusing
• Separation of proteins on basis of
isoelectric point
• Proteins migrate through pH
gradient until their overall charge
is neutral
• IEF strip soaked in buffer to impart
large negative charge to all
proteins (for next step)
Separation Techniques
2D-GE - Polyacrylamide Gel Electrophoresis
• Separation of proteins on basis of
size
• Small proteins migrate through gel
matrix quickest
• Resulting gel has proteins
separated
– Horizontally by IEP
– Vertically by size
Separation Techniques
2D-GE - Staining
• Proteins visualised by staining with
dyes or metals
• Different dyes have different
properties
– Silver stain
– Coomassie
– Fluorescent
Separation Techniques
2D-GE - Staining
QuickTime™ and a
decompressor
are needed to see this picture.
1ng
10ng 100ng 1000ng
Separation Techniques
2D Gel Electrophoresis
• Limitations
–
–
–
–
Resolution
Representation
Sensitivity
Reproducibility
• Advantages
– Established technology
• Still improving
– Quick
– Cheap (relatively)
Separation Techniques
DIGE
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Quic kT i me™ and a
dec om pres s or
are needed t o s ee thi s pi c ture.
QuickTi me™ and a
decompressor
are needed to see t his pict ure.
QuickT i me™ and a
decom pressor
are needed t o see thi s pi cture.
• DIfference Gel
Electrophoresis
• Variation of standard
2D-GE
– Multiple samples on
one gel
QuickTime™ and a
decompressor
are needed to see this picture.
Quic k Ti me™ and a
dec ompres s or
are needed to s ee t his pic t ure.
Quic k Ti me™ and a
dec ompres s or
are needed to s ee t his pic t ure.
• Usually 2 samples &
pooled reference
– Differentially labelled
– Eliminates running
differences between
gels
Separation Techniques
2D-GE Analysis
• Gel to Gel comparison identifies
varying protein spots
• Images overlaid and examined for
differences
• Relies on:
– Image warping
– Spot matching
– Quantitative spot volumes
Separation Techniques
2D-GE Analysis
• Progenesis SameSpots (Nonlinear
Dynamics)
• DeCyder (GE Healthcare)
• Delta2D (DeCodon GmBH)
Separation Techniques
Liquid Chromatography
• Proteins washed through capillary
column (or columns)
• Separates based on specific
properties
– Charge
– Size
– Hydrophobicity
• Depends on column matrix/eluent
Separation Techniques
Liquid Chromatography
• Usually 2 (or more) columns used
(MDLC)
• Can be coupled to Mass Spec (online)
• Or fractions collected for later
analysis (offline)
• Example: MudPIT (Multidimensional
Protein Identification Technology)
Separation Techniques
Liquid Chromatography
• Limitations
– No Peptide Mass Fingerprint
• Protein ID by MS/MS
– Expensive
– Difficult
• Advantages
–
–
–
–
Resolution
Representation
Sensitivity
Reproducibility
Separation Techniques
iTRAQ
Sample 1
digest
Sample 2
digest
+ Tag
Reporter
Moiety
+ Tag
N-hydroxy succinimide ester
for reaction with primary
amines (e.g. N-terminus of
peptides)
Balancer
Moiety
Total m/z of tag - 145
114
Calculate
abundance of
released
reporter
moiety
116
• Protein samples
digested and labelled
• Labels have different
MW reporters
• Differently labelled
peptides elute from
column together
• MS/MS allows
relative abundance
of 2 reporters to be
calculated
Separation Techniques
iTRAQ
Mass Spectrometry
The Basics
• Analytical technique that measures
Mass:Charge ratio (m/z) of ions
• Mass Spectrometers consist of 3 parts:
– An ion source
– A mass analyzer
– A detector system
• Only certain types of Mass Spec are used in
proteomics
– MALDI, SELDI or Electrospray ion sources
– Time of Flight, Quadrupole or Fourier Transform
mass analyzers
• Can Mass Spec whole proteins, but usually
just peptides
Mass Spectrometry
Ionisation - MALDI
• Matrix Assisted Laser Desorption/Ionisation
• Sample is mixed with matrix and allowed to
crystallise on a plate
• Laser fired at matrix (~100x) produces ions
• Typical matrix:
– 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic
acid)
– α-cyano-4-hydroxycinnamic acid (alpha-cyano or
alpha-matrix)
– 2,5-dihydroxybenzoic acid (DHB).
Mass Spectrometry
Ionisation - Electrospray (ESI)
•
•
•
•
Sample in volatile solvent
Introduced to highly charged needle
Forces charged droplets from needle
Solvent evaporation leaves only
charged sample
Mass Spectrometry
Mass Analysis - Time of Flight
• Ions mobilised by high voltage
• Travel through flight tube
• Deflected by reflectron (an ‘ion mirror’)
– Increases the path length (often doubles it)
– Therefore increases the resolution
• Time taken to reach detector is directly
proportional to mass of the analyte
Mass Spectrometry
Mass Analysis - Time of Flight
Mass Spectrometry
Mass Analysis - Quadrupole
• 2 different charges applied to 2 pairs of
metal rods
• Ions travel down the quadrupole
between the rods
• Only ions of a certain m/z will be able
to travel between the rods for a given
charge ratio
– Other ions will collide with the rods
• Spectrum produced by scanning
voltages
Mass Spectrometry
Mass Analysis - Quadrupole
Mass Spectrometry
Mass Analysis - Fourier Transform
• Fourier transform ion cyclotron
resonance
• Determines m/z based on cyclotron
frequency of ions in a fixed magnetic
field
• Ions do not hit the detector, but are
sensed as they pass close to it
• Produces a frequency spectrum
– A Fourier Transform procedure produces the
mass spectrum from this
Mass Spectrometry
Mass Analysis - Fourier Transform
Mass Spectrometry
Tandem MS
• Multiple mass analysis steps
• Separated by fragmentation
• Multiple methods of fragmenting
– collision-induced dissociation (CID)
– electron capture dissociation (ECD)
– electron transfer dissociation (ETD)
– chemically assisted fragmentation
(CAF)
Protein Identification
Peptide Mass Fingerprinting
• Proteases cut at defined sites
– e.g. trypsin cuts C-terminal of K or R
• Proteins cut with an enzyme will give a
series of peptides of different masses
• Different proteins will give different
series of peptides
• This is the peptide mass fingerprint of a
protein
Protein Identification
Peptide Mass Fingerprinting
• Alcohol dehydrogenase (374aa, human) gives
26 peptides greater than 500 Da
– 5795.795, 2861.4138, 2836.509, 2294.2069, 1685.9261, 1649.8493,
1645.8076, 1583.8315, 1557.7804, 1277.6228, 1181.7404, 1001.4833,
955.4731, 944.52, 920.5451, 889.4737, 885.5404, 846.4866, 827.4257,
780.4072, 695.2599, 648.3311, 622.3229, 580.3341, 573.2878, 564.281,
548.2787
• Guanine Nucleotide-Binding Protein, alpha-15
(374aa human) gives 31 peptides greater than
500 Da
– 3856.7945, 2092.0498, 1890.9748, 1864.0254, 1826.9734, 1769.8275,
1717.7924, 1690.8646, 1512.7263, 1360.6491, 1343.5606, 1326.5163,
1301.7212, 1295.6353, 1121.6565, 1083.6408, 1058.5339, 992.5299,
950.4434, 873.4424, 847.4407, 815.4621, 743.4661, 732.3522, 724.3876,
701.3253, 662.362, 660.3675, 595.345, 531.2885, 503.2936
• If you look at the two lists of peptide masses
you will not see any matches
Protein Identification
Peptide Mass Fingerprinting
• Alcohol dehydrogenase 7 (374 aa, human)
gives 26 peptides greater than 500 Da
– 5795.795, 2861.4138, 2836.509, 2294.2069, 1685.9261, 1649.8493,
1645.8076, 1583.8315, 1557.7804, 1277.6228, 1181.7404, 1001.4833,
955.4731, 944.52, 920.5451, 889.4737, 885.5404, 846.4866, 827.4257,
780.4072, 695.2599, 648.3311, 622.3229, 580.3341, 573.2878, 564.281,
548.2787
• Alcohol dehydrogenase beta2 (375 aa, human)
gives 25 peptides greater than 500 Da
– 4256.1078, 2846.4471, 2211.097, 1945.951, 1758.8003, 1729.9523,
1580.7261, 1555.8366, 1329.6797, 1202.6602, 1067.4826, 954.5982,
943.5094, 915.5298, 894.4753, 885.5404, 847.4268, 798.4144, 785.39,
637.3304, 594.2916, 580.3341, 543.3137, 526.2442, 516.2888
• Two closely related protein and yet only two
peptides match
Protein Identification
Peptide Mass Fingerprinting
699.45544, 896.32411, 909.51544, 909.75215,
912.58639, 920.50129, 973.56255, 1120.58328,
1127.71575, 1193.71203, 1508.56263, 1524.83725,
1525.14491, 1581.85175, 1718.0056, 1721.99879,
1979.20465, 2161.18785, 2184.04418, 2185.00575,
2201.3252, 2514.47913, 3354.92129, 3358.93766
QuickTime™ and a
decompressor
are needed to see this picture.
Deisotoping and
Noise Reduction
Extract
Peak List
Database
Search
QuickTime™ and a
decompressor
are needed to see this picture.
QuickTime™ and a
decompressor
are needed to see this picture.
Results
Protein Identification
MS/MS
• Peptides fragment in a predictable
way
• From an MS/MS spectrum, you can
work out the peptide sequence
• A peptide of >7 amino acids
should be sufficient to uniquely
identify a protein
Protein Identification
MS/MS
QuickTime™
and
a a
QuickTime™
and
decompressor
decompressor
areare
needed
to to
see
this
picture.
needed
see
this
picture.
Parent ion m/z = 1522.64
Daughter ion spectra can be deconvoluted to give sequence. The
major PMF search engines can also achieve protein ID by MS/MS
(MASCOT, SEAQUEST etc).
Role of Bioinformatics
• Software packages for image analysis
are complicated
– A large part of my job is training lab
biologists to use them
– Now moving into LC/MS analysis too
• Downstream analysis of experiments
– Similar in many ways to microarrays
– Visualisation of results can aid
understanding
• Data standards
– MIAPE, PSI, HUPO… more about this later
Summary
• Most proteomics experiments have
same skeleton
– Purification, Separation, Identification
• Many different technologies
– 2DGE, LC, MALDI, SELDI, TOF, FT etc
• Importance of bioinformatics
increasing
Any questions?
After the fact questions:
[email protected]