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Sensorer - Proteomics
Simon Ekström Department of Electrical Measurements/Create Health
Genomet
Genomet, vårt arv är lagrat i from av DNA
DNA är uppbyggt av 4 olika byggstenar ( A, T, G, C)
En 8-bitars sekvens DNA
kan kodas på 48 = 65538 sätt
Människan 5 x109 baspar
= 2 meter utvecklat
Duva ca 16 meter
Från gen till protein
Protein analys - Proteomik
•Proteom = den mängd olika proteiner som finns i en organism
•Proteinerna är uttrycket av vår genetiska potential
•Proteiner är involverade i alla biologiska processer
E. coli bakterie innehåller
ca 3000 olika proteiner
1. Human Genome Data – komplett, men ger inte svar på hur biologiska
processer fungerar.
2. Protein uttrycket i en organism är dynamiskt
3. 1 miljon olika proteiner i det humana proteomet?
4. Proteins finns i oilka koncentrationer 101-1012
5. Proteins modifieras ofta efter syntes
6. Ingen universell analys teknik
7. Proteiner antar tre-dimensionella strukturer som bestämmer funktioner
Protein problem
Cell Specific Expression of proteins
• All cells in a organism contain
the same genomic DNA
• But the genome do not answer the
question of why liver specific
genes are not expressed in brain?
• Drug discovery process begins with a
disease (rather than a treatment)
• Use disease model to pinpoint relevant
genetic/biological components (i.e.
possible drug target proteins)
Modern Drug Discovery
Relating druggable targets
to disease...
GPCR
STY kinases
Zinc peptidases
Serine
proteases
PDE
Other 110
families
Cys proteases
Gated ion-
channelIon channels
Nuclear
receptor
P450 enzymes
Analysis of Pharm industry reveals:
• Over 400 proteins used as drug targets
• Sequence analysis of these proteins shows that most targets fall within a few major gene families (GPCRs, kinases, proteases and peptidases)
Interesting facts...
• Over 90% of drugs entering clinical trials fail to make it to market
• The average cost to bring a new drug to market is estimated at $770 million
Fler proteiner observerade ---- men inte så många nya
Protein Assays !!!
Patient stratifiering
Domenici E, Wille R, Tozzi F, Prokopenko I, Miller S, McKeown A, Brittain C, Rujescu D, Giegling I, Turck CW, Holsboer
F, Bullmore ET, Middleton L, Merlo-Pich E, Alexander RC, Muglia P. (2010) Plasma protein biomarkers for depression
and schizophrenia by multi analyte profiling of case-control collections. PLoS One 5:e9166
Åldrande befolkning
Teknologier i life science
Proteomik Analys
What is Mass Spectrometry ?
A fancy word for a highly precise analytical balance!!!
– Analytical balances:
0.001g to 1g ± 0.0001g
– Mass spectrometers:
1e-24g to 1e-19g ± 1E-25g
Or
1 Da to 100.000 Da ± 0.1 Da
Basic Concept:
Play Ping-Pong with Molecules
• Accelerates and/or changes the trajectory of a charged particle by employing electric and magnetic fields and based on the observed behavior determines its m/z
• how much a particle responds to any outside electromagnetic field is determined by both its mass and charge – Higher mass => Less response
– Higher charge => More response
• m/z = 2m/2z , m/2z = 0.5m/z
2002 Nobel Prizes in
Chemistry
Mass spectrometry for
macromolecules "for their development of soft
desorption ionisation methods for
mass spectrometric analyses of
biological macromolecules"
Koichi Tanaka John B. Fenn
Mass spectrometry in proteomics as important as
PCR in Genomics!
How does a Mass Spectrometer work?
fundamental parts: the ionisation source, the analyser, the detector
MALDI
ESI
TOF, TQ,
QTOF, IT...
What sort of data can I get from MS?
Accurate molecular weight measurements:
purity of sample, detection of amino acid substitutions, post-
translational modifications, and disulphide bridges
Identification of individual compounds in mixtures
Sequence information
Reaction monitoring: enzyme activity, chemical modification…..
Protein structure:
protein folding (H/D exchange), protein-ligand complex
formation, macromolecular structure determination
Quantitaion: using isotope dilution
Not as sensitive as optical detection but provides an answer to
what analyte is detected
Where are MS applicable?
• Biotechnology:
analysis of proteins, peptides, oligonucleotides, polymers
• Pharmaceutical:
drug discovery, combinatorial chemistry, kinetics, metabolism
• Clinical:
neonatal screening, haemoglobin analysis, drug testing
• Environmental:
water, food, air quality (PCBs etc)
• Geological:
oil composition
Maldi vs ESI
• Off-line technique
• High through-put
• Medium information content
• Spot size is an important
parameter • Easy to use
• On-line best when interfaced
to LC separation
• Low through-put
• High information content
• Better MS/MS
MALDI for situations with many samples and lower complexity of samples
The two techniques are highly complementary
While MALDI approaches have a much higher potential for through-
put, sensitivity is limited by the lack of separation, lower dynamic
range and possibility for multiplexing
Schematic of ESI MS developed by John Fenn (taken from Fenn’s Nobel Prize lecture)
Electrospray ionization (ESI MS)
MALDI: Matrix Assisted Laser Desorption
Ionization
hn
Laser
1. Sample (A) is mixed with
excess matrix (M) and dried
on a MALDI plate.
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are ionized
by proton transfer from matrix:
MH+ + A M + AH+.
AH+
+20 kV
Variable Ground
Grid Grid
Sample plate
Time-of-flight mass analyzer
+
+
+
+
Source Drift region (flight tube)
dete
cto
r
V
•Ions are formed in pulses.
•Small ions reach the detector before large ones.
•Measures the time for ions to reach the detector.
Calibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the square
of its drift time.
t = Drift time
L = Drift length
m = Mass
K = Kinetic energy of ion
z = Number of charges on ion
2
22
L
Kt
z
m
Typical MALDI TOF
Camera
Laser
Sample
plate
Pumping Pumping
Timed ion selector Reflector
Linear
detector Extraction
grids Reflector
detector Attenuator
Prism
Collision
cell
+ e -
primary ion
e -
e - e - L
D
- 1000V
- 100V
L >> D
Ions are detected with a microchannel plate
The problem: Peaks are inherently broad in MALDI-TOF
spectra (poor mass resolution).
+ +
+
Sample + matrix on target
Ions of same mass, different velocities
The cause: Ions of the same mass coming from the target
have different speeds. This is due to uneven energy
distribution when the ions are formed by the laser pulse.
Step 1: No applied electric field. Ions spread out.
+
+ +
Ions of same mass, different
velocities
Step 2: Field applied. Slow ions accelerated more than fast ones.
0 V.
0 V.
+
+ +
Step 3: Slow ions catch up with faster ones.
20 kV.
20 kV.
0 V.
0 V.
+
+ +
Delayed Extraction (DE)
improves performance
0 V. +20 kV
A reflector focuses ions to
give better mass resolution
+
+
Reflector
Resolution
Ability of a mass spectrometer to distinguish between ions of different m/z ratios. R=m/Δm
Δm is the width of the peak at half maxima (FWHM) of the peak corresponding to m.
If we have 5000 resolution on a mass spectrometer, we can separate m/z
50.000 from m/z 50.010,
Resolution & mass accuracy on mellitin
0
2000
4000
6000
8000
Counts
2840 2845 2850 2855
Mass (m/z)
Resolution = 14200
Resolution = 4500
Resolution = 18100 15 ppm error
24 ppm error
55 ppm error
MS-MS, SIM and SRM possible
Protein identification – by
PMF
Analytical Approach to Peptide Mass Fingerprinting:
Effect of Mass Tolerance
1529 1 478
1529.7 0.1 164
1529.73 0.01 25
1529.734 0.001 4
1529.7348 0.0001 2
Search m/z Mass Tolerance (Da) # Hits Database
But if we use MS/MS and get some sequence e.g a peptide
1529.7348 with the sequence RYIXXXX it improves futher
Tandem MS (MS/MS) gives sequence
A first mass spectrometer (MS1) is used to SELECT, from the
primary ions, those of a particular m/z value which then pass into
a Fragmentation Region. The daughter ions are analysed in the
Second Spectrometer (MS2). In fact, the MS1 can be viewed as
an ion source for MS2.
Quantitative MALDI MS
analysis IS as for ESI, but lower mutiplexing capability in maldi and worse label free
MALDI ion suppresion
A quantitative problem as it is very unpredictable
Investigating the Quantitative Nature of MALDI-TOF MS
http://www.mcponline.org/content/7/12/2410.full
MALDI sweet spots/hot spots
MALDI signal is rarely the same over the spot area
Many methods for reducing spot size; hydrophobic targets,
Electrospraying, dispensing, SAW , etc……
Posttranslational Modifications Intra- versus intermolecular disulfide bridges
S S
SH
E E
protein
S S
SH
peptide mixture
cleavage SH
HS
SH
peptide mixture
reduction
m/z
inte
sity
MALDI MS
m/z
inte
sity
MALDI MS
Posttranslational Modifications
Phosporylation – identification of peptides
E E
PO3
PO3
cleavage
PO3
PO3
Dm = 98 Dm = 98
m/z
inte
sity
linear MALDI MS
m/z
inte
sity
reflectron MALDI MS
MALDI Screening
applications
Ratio substrate:product
PKA
Mass Spectrometry Reviews, 2007, 26, 324– 339
Advances in Bacterial Identification
• Most significant advance in Clinical Microbiology (Bacteriology) in 30
years!
– Rapid and cost effective identification of bacteria directly from
isolated colonies and positive culture bottles based on protein
biomarkers
• Protein biomarkers measured are highly expressed proteins
responsible for housekeeping functions, such as ribosomal
(16S) and transcription/translation factor proteins
Biochemical to MALDI-TOF Bacterial
Identification
Conventional ID vs MALDI
• Monday, 12pm, Mr. J’s blood culture flags positive
• Bottle removed, gram stain /culture prepared
• Gram negative rods seen, floor called at 1:10pm
• 3pm – Mr. J started on Ceftriaxone
• Tuesday, 10:30am P. aeruginosa identified
• Floor called 10:45am
• Mr J started on Pip/tazo
• MALDI ID would have seen Mr J on appropriate
therapy 20-24 hours earlier
Add matrix solution*
Air dry for 1-2 min.
MALDI TOF Sample Preparation
Create Spectra
Target Slide
48 wells
Step 1 Step 2 Step 3 Step 4
Bacteria, molds, yeasts,
Mycobacteria
Spot target slide with direct colony (can be
up to 5 days old).
Load target slides
NOTE: Other sample types: - sediment from positve blood cultures - sediment from certain specimen (e.g. urines)
47
Direct Detection for Positive Blood Culture Bottles By MALDI
Purpose: Separate human and bacterial/yeast ribosomal proteins Methods: Lysis/centrifugation or membrane filtration
Journal of Clinical Microbiology 51;805-809, 2013
Journal of Clinical Microbiology 48;1584-1591, 2010
Issues:
• Removal of human proteins
• Extraction protocol required
• Bacterial concentration
• need~107/mL
• Polymicrobial specimens
• Seen on Gram stain?
• Charcoal
• Antibiotic resistance genes
• Yeasts?
• Unique database, different cutoffs?
Acoustic Trapping for Bacteria Identification in
Positive Blood Cultures with MALDI-TOF MS
Anal. Chem., 2014, 86 (21), pp 10560–10567
Slice frozen embedded
tissue on cryostat
at 10 μm
Serial sections acquired
Spray
with matrix
Acquire
mass spectra
TestWCXreflectronQC\0_C4\1\1SRef
0.0
0.5
1.0
1.5
2.0
4x10
Inte
ns.
[a.u
.]
500 1000 1500 2000 2500 3000 3500m/zMolecular profile
Molecular image
Adjacent section H & E stained
Compare molecular image to stained
serial section
conductive
slide for
MALDI-MSI Pathologist defines
areas of interest
ImagePrep
UltraFlex III TOF/TOF
w/ Smartbeam
Mass spectrometry imaging - MALDI-MSI
PCa PCa
Benign
stroma
0
100
Benign
4000 6000 8000 10000 12000 14000 16000 18000
Rela
tive I
nte
nsity
Mass to charge (m/z)
stroma
m/z 4355
Tumor
Benign
2mm
MALDI-IMS Utilizing m/z 4355 can Identify
PCa Specific Regions of Prostate
MALDI imaging of m/z 4355 Histology
Cazares, L.H., Troyer, D.A., et al. (2009)Clin. Cancer Res., 15, 5541-5551.
SELDI ???
Fluorescence
Confocal analysis of single molecular
events is accomplished using confocal
optics with an illumination/detection
volume of -1 fl
FRET
no FRET
FRET (fluorescense resonance energy
transfer) describes an energy transfer
mechanism between two chromophores.
(good for reaction monitoring)
Array-based fluorescence
detection of biomolecules
Surface area, chemistry and deposition
technique important
Antibody assays
Are proteins used by the
immune system to identify and
neutralize foreign objects, such
as bacteria and viruses.
produced by a kind of white
blood cell called B cells.
Direct assay
Sandwich
assay
Antibody based tests Pregnancy - Human chorionic gonadotropin (hCG)
10 - 200 sek
Lab on a chip / Point of care
POC test
[Glucose, HbA1C, microalbumin, electrolytes, cholesterol, C-
reactive protein, urinalysis, chlamydia, HIV, coagulation
markers, streptococcal infection, blood gases, creatinine,
amylase, drugs (overdose and abuse), cardiac markers,
brain-specific proteins,