MS-IMS (MALDI-IMAGING)?

Protein Chemistry/Proteomics and Peptide Synthesis and Array UnitBiomedicum Helsinki and Haartman Institute

E-Mail: marc.baumann@helsinki.fi

(http://research.med.helsinki.fi/corefacilities/proteinchem)

MALDI mass spectrometric imaging of biological tissue sectionssections

Tatiana C Rohner Dieter Staab and Markus StoeckliTatiana C. Rohner, Dieter Staab and Markus StoeckliNovartis Institutes for BioMedical Research, Novartis Pharma AG, Lichtstrasse 35, CH-4002 Basel, Switzerland

Mechanisms of Ageing and Development Mechanisms of Ageing and Development Volume 126, Issue 1 , January 2005, Pages 177-185

Image (tissue, cell, bacteria, virus ) Scanning with a massvirus...) Scanning with a mass

spectrometer

Definition

MS is an analysis technique to determine by means of a mass spectrometer the molecule

mass of free ions in high vacuum

MALDI Principles

• Mix analytes with excess matrix compound lli (1 1000 10 000)to crystallize (1:1000 or 10 000)

• Matrix absorbs at the same laser wave length (commercially available N2-g ( ylaser@337nm, YAG laser @355nm, infrared Er-YAG@2.94um)@ )

• Short waved laser pulses• Short waved laser pulses

MALDI-TOF/TOF for LID-LIFT and high energy CID-LIFT

LIFT Precursor Ion Selector

CID cellTarget CID cell(e.g. Argon)

Potential Profile during LIFT

LIFT

Tissue slide for IMS-MS

100µm raster step

• Definitions:e t o s:

• MALDI Imaging (MALDI-IMS)(MS-IMS)g g ( )( )

• A technique for analyzing the spatial arrangement of proteins, peptides, lipids, and small molecules in biological tissues

• A protein profiling technique which enables the direct sampling of histological section

• A technology that utilizes MALDI MS to map molecules of interest in thin tissue sections

• Potentially can deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatmentof these ions during development and disease progression or treatment

PrinciplesTissue section (mouse brain)

on y

Acq

uisi

tio

Acquisition x

12mm

Ion

inte

nsity

2,000 15,000 30,000

m/z

• A laser is rastered over a defined area while acquiring a complete mass spectrum from each positionA laser is rastered over a defined area while acquiring a complete mass spectrum from each position, resulting in molecular images for multiple analytesCornett, et al., Nature Methods 2007

IMS workflow

Stoeckli, M, et al., Analytical Biochemistry 2002

Benefits of MALDI-MSI

• Analysis of entire sample in one readingsample in one reading

P i k l d• Previous knowledge of molecular composition is not necessarynecessary

All f• Allows for investigation of disease formation, progression andprogression, and treatment

www.maldi-msi.org

MS i iTargeted labeling

MS imaging advantages

•No labeling requiredBiomolecules are functionally yunmodified

•Image biomolecular Label free imagingmodificationsPTM’s, Metabolites

D il d i f i•Detailed information on molecular identity•Large scope of different elementsdifferent elements and molecules

IMS vs. histochemical stain

Imaging MS applications……

Drug administration and detection

New Phytologist (2007) 173 : 438 –444m/z 381

m/z 543

m/z 705

Reconstruction of the Carbohydrate moietyOf WHEAT (Triticum aestivum)

Collection and storage of samples

Tissue preparation and matrixEmbedding applicationEmbedding

Section mounting

Heeren RMA, USAFixed versus fresh tissue

Tissue preparation and matrixapplication

Heating 1-5 min

Rapid Heating=treatment untreated Slow warming

Sample pretreatment prior to matrix application

Tissue thickness 10-20 um

On tissue digestion (spot and spray)

2D- IMS-MS for protein identification

Peptide digestion on the membrane (identification of the proteins)Peptide digestion on the membrane (identification of the proteins)

Direct trypsin spraying; (Jardin-Mathé et al., 2008)

Comparing Regions of interestp g g

Control vs. patient

Matrix application methodsMatrix application methods

Matrix Application

• Matrix application is vital for quality image resolutionresolution

• Must contact sample as fine, liquid mist

• Current procedure• Current procedure involves manual application with airbrush 100 t tairbrush 100µm raster step

Barrett-Wilt, G., USA

Home made spray device

1. Automatic Spray Gun

2 Conveyor1

4

2. Conveyor

3. DC Timing Motor

24. Integrated Polyethylene Box

3

2

3

www.buswire.ocr.wisc.edu

Bruker IMAGE-PREPWith SPR

Nano spotter

Special ApplicationsSpecial Applications

Immuno-MS-IMS

Imaging of regions immunoreactive with anti-synaptophysin Ab in healthy human pancreas.

(A) Localization of synaptophysin positive cells by TAMSIM. The monoclonal rabbit anti-synaptophysin is conjugated with the tag El 307 (498 m/z). The false color green points in the section show the presence of the tag El 307 and thus synaptophysin positive cells.

(B) Classical IHC image with the anti-insulin Ab. The dark pink spots correspond to Langerhans islets and so the synaptophysin-positive cells. The distribution of synaptophysin positive cells in (A) is very similar to that in (B)that in (B).

Thiery, G., et al., Proteomics 2008

Whole animal IMS-MS

Effect of rasteringg

Microchips on IMSMicrochips on IMS

Imaging MS for studying analyte deposition in dried droplet methodsp

•Distribution of analytes / particles in dried droplet deposition is not even.

• Typical case: stronger deposition atTypical case: stronger deposition at edges (coffee droplet effect).

• Matrix-free ionization of peptides.

1 mm circle and square shaped spots

Raster spacing 25 µm or 50 µmaste spac g 5 µ o 50 µ

Some preliminary results

Angiotensin 2, 1 µl, 1 pmol

1 mm spot [A+H]+1 mm spot [A+H]

2 mm spot [A+H]+

2 mm spot [A+K]+p [ ]

Some preliminary results

Angiotensin 2, 1 µl, 1 pmol

1 mm spot [A+H]+1 mm spot [A+H]

2 mm spot [A+H]+

Divider/concentrator spotp

Bi3+ ion imaging of peptide array on ion-NIMS surface

Other use of...

- Laser Capture MC of single cells- protein profiling directly from gelsetc

1-DE Chip electrodes

sample inlet

gel reservoir: water coolinggel reservoir

sample inletbuffer reservoir

25x32x0.3/0.5 mm PMMA, silica

standard

real sample

Running time 10 minutes

2-DE Chip

T�� �������� ������

�������� 2�DE ������

• PerformanceHbA1cHbF

HbA2

Native IEF and native PAGE

5 variants of hemoglobin

pH 6.7 -7.7

HbA1c

HbA

HbF

HbS

p

Native IEF and SDS-PAGE

standard IEF proteins

pH 3-10

Denatured IEF and SDS-PAGE

GFAP protein variants expression differences

in control and Alzheimer diseased patients

pH 4-6

control AD

DIOS-MS

Desorption Ionization On Silicon (DIOS)Matrix Free IMS

laser MS

porous area sample

3D IMS-MS3D IMS MS

Resolving power of an MS

IMS FT ICR MSIMS FT-ICR-MS(Fourier Transformation Ion Cyclotron Resonance MS)

Sensitivity in the zmol range

IMS-MS combined with other technologiesIMS MS combined with other technologies