Matrix-assisted LaserDesorption/Ionization

(MALDI) -ImagingDiane Hatziioanou (Άρτεμις)

Postdoctoral Researcher

• MALDI-Imaging Workflow • How MALDI-Imaging works• MALDI-Imaging data information• Preliminary results• Comments on 2D protein electrophoresis

MALDI-Imaging Workflow

Workflow Tissue preparation

•Tissue is snap frozen in liquid N2

Image from Wikipedia

Workflow Tissue preparation

•Embedding material such as OCT is avoided as these polymers suppress ion signals and create background signals in MALDI-MS

Embedded in OCT

No OCT

Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

Workflow Slide preparation

•Tissue is cut using a cryostat into 5-15μm sections•Tissue sections are thaw mounted onto ITO-coated slides

Workflow Slide treatment

• Slides are:• Desiccated to remove moisture• Washed in Ethanol to remove salts and

contaminants• Optionally washed in organic solvents to

remove lipids• Scanned/imaged• Coated with a matrix

RatKidney

Workflow Slide Imaging/Scanning

• Slides are:• Scanned using a conventional scanner• Photographed from a microscope (and patched

together)

• Scanned using an aperiscope

RatKidney

Workflow Matrix selection

• SA routinely used for higher molecular weight proteins• SA yields the best combination

of crystal coverage and signal quality.

• CHCA used for lower MW peptide species

• DHB used for both mass ranges in a single experiment

Workflow Matrix concentration selection

Effect of matrix concentration on crystallization and the resulting mass spectra.

Solutions of SA in 50 : 50 acetonitrile/0.1% TFA

(A) 10 mg/ml

(B) 20 mg/ml

(C) saturated (>30 mg/ml)

Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

Workflow Matrix solution selection

• Water and organic solvent mixture allows both hydrophobic and water-soluble (hydrophilic) molecules to dissolve into the solution

• Acetone• Methanol• Isopropanol• Acetonitrile• Ethanol

• The liquids vaporize, leaving co-crystallized matrix with analyte molecules. • Co-crystallization is a key issue in selecting a proper matrix to obtain a good

quality mass spectrum of the analyte of interest

• Sample acidification with up to 0.2% TFA may improve spectra• Detergents (eg 0.05% v/v Triton X-100 or SDS) may improve detection of membrane proteins,

hydrophobic proteins and also increase overall protein signal intensities

Mainini V, Angel PM, Magni F, Caprioli RM. Rapid Commun Mass Spectrom. 2011 Jan 15;25(1):199-204. doi: 10.1002/rcm.4850.

Workflow Matrix solution selection

Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

Workflow Matrix solution selection –TFA concentration

Schwartz, Sarah A., Michelle L. Reyzer, and Richard M. Caprioli. Journal of Mass Spectrometry 38.7 (2003): 699-708.

Workflow Detergent effect on Matrix

Mainini V, Angel PM, Magni F, Caprioli RM. Rapid Commun Mass Spectrom. 2011 Jan 15;25(1):199-204. doi: 10.1002/rcm.4850.

Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016

Workflow Solvent/TFA effect on Matrix

Workflow Solvent/TFA effect on Matrix

Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016

AcN

EtOH

MeOH

Workflow Matrix solution Application Method

• Spotting• Manual deposition• CHIP-1000 spotting device (piezoelectric

technology) -200 μm• Coating (delivery of a homogeneous layer of matrix over the entire tissue)

• Imageprep (vibrational vaporization) -50 μm• SunCollect sprayer(pneumatic sprayer) -50 μm• Thin layer chromatography (TLC) sprayer

• Two-step approaches: • Sublimation followed by recrystallization 1-2 μm

Workflow MALDI-Imaging using a Bruker autoflex speed

• Slides are inserted into the Imager• Software settings are selected and a

scanned slide image is calibrated to match the orientation of the imaged slide.

• Protein standards, parts of the test tissue and surrounding matrix are hit with the laser to test its performance.

Live video of slide

Grid for positioning laser/video

Laser results (Calibration protein)

Software settings

Workflow MALDI-Imaging using a Bruker autoflex speed –part 2

• An area of tissue is selected for Imaging

• Several areas can be selected from up to 2 slides

• Imaging of the selected areas is performed (1h –o/n)

• Imaging data can be viewed as average spectra, average spectra of sub-areas or distribution of single mass peaks.

How MALDI-Imaging works

MALDI is a two-step process– A UV laser beam triggers desorption. • Matrix material absorbs UV laser light and the upper

layer (~1 μm) of the matrix material is ablated– The ablated plume contains many species: neutral and ionized

matrix molecules, protonated and deprotonated matrix molecules, matrix clusters and nanodroplets.

– Analyte molecules are ionized (protonated or deprotonated) in the hot plume

– eg. [M+H]+ (added proton), [M+Na]+ (added sodium ion), [M-H]- (removed proton)

Laser

Ionized analytes

Laser

Ionized analytes

J. Kathleen Lewis, Jing Wei, Gary Siuzdak, Peptides and Proteins 2006 DOI: 10.1002/9780470027318.a1621

Laser

Ionized analytes

Hillenkamp, Franz, and Jasna Peter-Katalinic, eds. MALDI MS. John Wiley & Sons, 2007.

High-speed time-lapse photographsof IR-MALDI plumes with 100-ns pulse width Matrix: glycerol; timeresolution 8 ns; spatial resolution 4μm.

Separation and detection of MALDI ionized analytes (Mass spectrometry)

Ions’ Time Of Flight (TOF) analysis• Ions are accelerated to a detector• The arrival time at the detector is dependent

upon the mass, charge, and kinetic energy (KE) of the ion. – KE is equal to ½ mv2 (where v=velocity). Ions will

travel a given distance, d, within a time, t, where t is dependent upon their mass-to-charge ratio (m/z)

– Increased resolution often comes at the expense of sensitivity and a relatively low mass range(< 10 000 m /z)

Mass spectrometry -TOF Analyser• Reflectors increase the mount of time (t) ions need to reach the detector while reducing

their KE distribution, thereby reducing the temporal distribution Δt. • Resolution is defined by “peak mass” divided by “peak width” m /Δm (or t/ΔT).

Increasing t and decreasing Δt results in higher resolution.

• Once the mass spectrum is acquired the sample is moved by a defines distance and the next position in the sample is analyzed the same way.

J. Kathleen Lewis, Jing Wei, Gary Siuzdak, Peptides and Proteins 2006 DOI: 10.1002/9780470027318.a1621

MALDI analyzers• MALDI MS is

– most commonly combined with TOF mass analyzers.– MALDI MS can alternatively be combined with Ultrahigh-resolution ( > 105) mass

analyzers such as the Fourier transform ion cyclotron resonance (ICR) mass analyzer – called Fourier transform mass spectrometry (FTMS) .

Analyzer

Meyers, Robert A., ed. "Encyclopedia of analytical chemistry." (2000).

MALDI-Imaging data information

MALDI-Imaging data information

MALDI-Imaging data can give spatial distribution patterns even at 200 μm resolution

Bruker Daltonics Application Note # MT-91

Whole-organ MALDI Imaging

Whole-Animal MALDI ImaginUninfectedInfected

Ahmed S. Attia,, et . al. Monitoring the Inflammatory Response to Infection through the Integration of MALDI IMS and MRI, Cell Host & Microbe, 2012 (11) 664-73

H&E-stained sections of entire mice

Masses corresponding to proteins that are abundant in the liver (m/z 3,562), kidney (m/z 5,020), brain (m/z 10,258), or systemically (m/z 11,837) in both infected and uninfected mice are shown.

In addition, masses corresponding to proteins that are only expressed in infected animals are shown (m/z 10,165, 10,202, 10,369).

Separation and detection in MALDI-Imaging

• The 3D structure of the samples affects ion flight times and results in significantly lower mass resolution and mass accuracy

• Mass deviations up to 0.5 m/z not uncommon.• Spatial resolution typically 50-200 μm per pixel.

– Resolution up to 1 μm possible.

• Samples can be trypsin digested to detect larger molecules– Matches with LC-ESI-MS/MS only possible with low ppm range mass

accuracy for both measurement models, less accurate measurements lead to ambiguous assignments.

Römpp A, Spengler B. Histochem Cell Biol. 2013 Jun;139(6):759-83. doi: 10.1007/s00418-013-1097-6.

MALDI-Imaging Drawbacks

• Only detects the most abundant molecules • Difficult to detect proteins over 20 kDa• Identification of masses possible only with low

ppm range mass accuracy less accurate measurements lead to ambiguous assignments.– But... Results and profile publishable without

identification.

MALDI-Imaging Benefits

• Spatial profiling• Analysis of all parts of sample in one reading• Untargeted (label free), multiplex method. – Add desorbed and ionized compounds in the sample are

detected, regardless whether known/unknown/expected /unexpected

• Can optimize conditions to detect proteins, peptides, lipids, drug compounds a.o.

• Allows for investigation of disease formation, progression, and treatment

Preliminary work

MALDI-Imaging work

• 2 MALDI-Imaging slides run– Cryosectioning training obtained– Instrument time and supervision not always

available• Conditions used were those used in lab for

brain tissue.– Imageprep used for matrix deposition– Insufficient amount of matrix used.

MALDI-Imaging workNo PBS1

PBS wash

(Background)

+35mg/ml SA in 50:50 AcN

10mg/ml SA in 60:40 AcN, 0.2% TFA

MALDI-Imaging work-Images from kidney taken from a dead rat.

30mg/ml SA in 70:30 AcN, 0.1% TFA

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Control

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Comments on 2D protein electrophoresis

2D protein electrophoresis work

• Optimized conditions work very well• Results (24 samples -without data analysis)

deliverable within 1-2 months

2D protein electrophoresis work

12% SDS-PAGE

15--kDa

180-kDa

10% SDS-PAGE15--kDa

180-kDa

11% SDS-PAGE

15--kDa

180-kDa

National and Kapodistrian University of Athens

• Vlahakos Dimitrios

BRFAA• Charonis & lab

– George Barkas

• Vlahou & lab– Manousos Klados– Makis Zoidakis– Vasiliki Bitsika

Demokritos• Tsilibary & lab

– Aspasia Volakaki

National and Kapodistrian University of Athens

Università degli Studi di Milano-Bicocca

• Magni & lab– Andrew Smith

Many thanks to:

Sublimation Device

Joseph A. Hankin, Robert M. Barkley, and Robert C. Murphy J Am Soc Mass Spectrom. Sep 2007; 18(9): 1646–1652.

UV MALDI Matrix ListCompound Other Names Solvent Wavelength

(nm) Applications

2,5-dihydroxy benzoic acid[1]

DHB, Gentisic acid

acetonitrile, water, methanol, acetone,

chloroform 337, 355, 266

peptides, nucleotides, oligonucleotides, oligosaccharides

3,5-dimethoxy-4-hydroxycinnamic acid[2]

[3]

sinapic acid; sinapinic acid;

SA

acetonitrile, water, acetone,

chloroform 337, 355, 266peptides, proteins,

lipids4-hydroxy-3-

methoxycinnamic acid[2][3] ferulic acid

acetonitrile, water, propanol 337, 355, 266 proteins

α-Cyano-4-hydroxycinnamic acid[4]

CHCAacetonitrile, water, ethanol, acetone 337, 355

peptides, lipids, nucleotides

Picolinic acid[5] PA Ethanol 266 oligonucleotides3-hydroxy picolinic

acid[6] HPA Ethanol 337, 355 oligonucleotides

Workflow Solvent/TFA effect on Matrix

Cassie Gregson. (2009). Optimization of MALDI tissue imaging and correlation with immunohistochemistry in rat kidney sections. Bioscience Horizons. doi:10.1093/biohorizons/hzp016

AcN

EtOH

MeOH

MALDI images and spectra rat kidney sections (A) Male 3, (B) Male 5, (C) Female 2, (D) Female 5 (E) intensity legend, where: (i) image at m/z 15.3 with spectra from area highlighted within

tissue section, (ii) spectrum from outside of the tissue boundaries and (iii) image and spectrum at m/z 18.7.

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