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,