Introduction

The molecular mechanisms involved in most

neurodegenerative disorders, such as Parkinson’s

and Alzheimer’s disease, are still unclear. Most

protein expression analyses have been performed

on homogenized preparations of whole tissues.

Such preparations do not provide any information

about relevant changes in specific cell types. The

aim of this study was to examine whether laser-

microdissected samples of single cell types from

post-mortem brain tissue can be used for protein

expression analysis.

Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapultand Nano-LC/MS/MS

Application Note

Clinical ResearchChristian SauberAgilent Technologies

Bernd SägmüllerPALM Microlaser Technologies

Manuela Neumann and Hans A. KretschmarLudwig-Maximilians-Universität München

Experimental

Sample preparation

The tissue samples were obtained from theDepartment of Neuropathology, Brain-Bank,Ludwig-Maximilians-Universität in Munich, Ger-many. The samples, taken from post-mortemhuman cerebellum and snap frozen, containedportions of white matter and cortex. Sampleswere used as provided by neuropathology at athickness of 10–15 µm, representing a cell layer.The staining procedure included a standard hem-atoxylin-eosin (HE) staining protocol (see Figure 1).

Laser microdissection and pressure catapulting(LMPC) of human brain tissue was carried outusing a PALM MicroBeam IP system featuring a337-nm nitrogen laser. Software control of thelaser pressure catapulting (LPC) and homogeniza-tion was provided by the AutoLPC function in thePALM RoboSoftware. Sample collection was per-formed by outlining the regions of interest in

Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

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Tryptic digestion took place at 37°C overnight at aconcentration of 800 ng trypsin for 10 mm2 cellarea, i.e. a ratio of 1:20 for protease : protein.After stopping the reaction with 1% formic acid,the sample was lyophilized. It was reconstitutedin 20 µl of 0.1% formic acid in water for subse-quent LC/MS analysis.

Digestion protocol for TFE/trypsinSamples were catapulted directly into 15 µL of asolution of 50% TFE (1,1,1-trifluoroethanol) and50% 30 mM ammonium bicarbonate in water with 10 mM DTT (pH about 8). Samples were also cata-pulted into PALM AdhesiveCaps for prolonged cellharvest and then denaturated afterwards.

After centrifugation, the sample was held at 60°C for 45 minutes. The sample was diluted with 90 µL of 30 mM ammonium bicarbonate buffer.Trypsin was added as described in the tris/ureaprotocol. After stopping the reaction with 1%formic acid, the sample was lyophilized. It wasreconstituted in 20 µL 0.1% formic acid in waterfor subsequent LC/MS analysis.

LC/MS analysis

All LC/MS experiments were performed using anAgilent Nanoflow Proteomics Solution featuringan Agilent 1100 Series nano-LC for MS coupledthrough an orthogonal nanospray ion source to anAgilent 1100 Series LC/MSD Trap XCT ion trapmass spectrometer. The nano-LC system was oper-ated in sample enrichment/desalting mode using a ZORBAX 300SB-C18 enrichment column (0.3 ×50 mm, 5 µm). Chromatography was performedusing a ZORBAX 300 SB-C18 (75 µm × 150 mm)nanocolumn. The solvent gradient started at 3%solvent B (0.1% formic acid in acetonitrile) and97% solvent A (0.1% formic acid in water).

The LC/MSD Trap XCT was operated in theunique peptide scan auto-MS/MS mode. Peptidescan mode acquires full-scan MS spectra at a scanspeed of 8100 u per sec and a resolution of less

Agilent Technologies

areas adding up to 5, 10 and 20 mm2. The stepwidthof the AutoLPC grid was set to 5 µm, assuring each cell was pulsed at least two times for reliablehomogenization.

The cellular fragments were collected directly inthe caps of microfuge tubes. Before LMPC, a drop(15 µl) of the denaturation liquid was placed inthe center of each cap. The surface tension of thedenaturation liquid allowed droplets of this size tobe safely used upside-down. The caps were placedabove the sample and as close as possible to thefocal volume of the laser. Tissue catapulted by thelaser landed in the denaturation liquid.

After LPC, the tissue sample was immediatelydigested using two different protocols: one astandard urea protocol, the other a mixed organicsolvent protocol.

Digestion

Digestion protocol for urea/trypsinSamples were catapulted directly into 15 µl Tris/6 M urea buffer in the cap for instant denatura-tion or into PALM AdhesiveCaps for prolonged cellharvest and then later denaturing. After an hourthe suspensions were diluted by adding 95 µlwater and then vortexing gently.

Figure 1. Image of the cerebellum tissue sample. On the left isthe white matter, on the right cortex. The cortex structure isenhanced by blue color.

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Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

Agilent Technologies

INSTRUMENT CONDITIONS

LC Conditions:

Enrichment

Column: Zorbax SB-C18, 300A, 0.3 x 5 mm, 5 µm

Mobile phase: 0.1% formic acid in water

Flow rate: 10 µL/min

Analysis

Column: Zorbax SB-C18, 300A, 0.075 x 150 mm, 3.5 µm

Mobile phase: A: 0.1% formic acid in water

B: 0.1% formic acid in acetonitrile (ACN)

Gradient: 0-5 min at 3%B

then 0.5%B per minute up to 60% (5–119min)

60% to 85%B over one minute (119–120min)

held at 85%B

stop time 125min

Flow rate: 250 nL/min

Injection: 16 µL out of 20 µL

MS Conditions

Ionization mode: Positive nanoelectrospray

Drying gas flow: 5 L/min

Drying gas temperature: 300°C

Skimmer: 30 V

Capillary exit: 75 V

Trap Drive: 85

ICC: On

Peptide scan mode

Active exclusion: 3 spectra, 1.5 min

Prefered charge state: Doubly charged

Number of precursors: 2

Maximum accumulation 50 ms

time:

Target: 125,000 (MS)

100,000 (MS/MS)

Scan: 300 – 2200 m/z (MS)

100 – 1800 m/z (MS/MS)

than 0.35 u (FWHM). It uses automated, data-dependent MS/MS to acquire MS/MS spectra at26,000 u per sec and a resolution of less than 0.6u (FWHM) from the two most abundant peaks inthe MS spectrum. Peptide scan mode allows forcharge-state determination based on isotopic res-olution up to +3 with an overall acquisition rateof 1 MS/MS spectrum per second.

Data processing

All acquired data were processed using theAgilent Spectrum Mill MS proteomics workbenchto search the NCBI database (Human). Spectralpreprocessing was performed by the SpectrumMill extractor program. Only MS/MS spectra witha sequence tag length greater than 1 were selectedas potential peptide spectra for the database search.

Results and Discussion

In contrast to RNA analysis of LMPC tissue (seeAgilent publication number 5988-9128EN), pro-teins from tissue cannot be amplified to improvedetection limits. Therefore it was decided to workon larger tissue areas between 1 and 20 mm2

where 105 cells are assumed at 10 mm2. Each cellcontributed about 4 ng of protein, leading to some400 µg of protein in total for protein profiling.

During the LMPC process, the denaturation liquidevaporated to a negligible extent in the case of acell harvest from up to 5 mm2 area. However, afterthe collection was complete, an additional volumeof some 10 µl was used to assure the completeimmersion of all flakes present in the denatura-tion liquid.

The prolonged harvesting of cells (10 and 20 mm2),which led to the best results in protein profiling,was obtained using PALM AdhesiveCaps. Thesecaps provided a filling that allows specimen col-lection without the need to place buffer in thecap. The denaturation buffer was applied afterthe cell collection was finished, followed by abrief vortexing and a spin-down of the denatura-tion liquid and the cell-flakes.

Agilent Technologies Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

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Glass slidewith specimen

Microscopeobjective

Cap with droplet ofdenaturation buffer

Figure 2. Schematicrepresentation of theLMPC setup

Figure 3. LMPC-homogenized flakes found in the cap withdenaturation buffer

Major benefits of using laser pressure catapultingcompared to traditional glassware homogenizedtissue are that a well-defined tissue area or evenseveral areas can be harvested without touchingthe sample. This process can be carefully moni-tored and documented with a built-in microscope.Through the AutoLPC capability of the MicroBeaminstrument, the individual cells were already laserpulse homogenized during the catapulting processand soluble proteins were directly accessible forfurther treatment (Figures 2, 3)

Two different digestion protocols were comparedto evaluate the impact on peptide/protein identifi-cation. Complex protein mixtures from tissuerequire a dedicated procedure to guarantee com-plete digestion of the proteins. The protocol usinga mixed-organic solvent system resulted in morepeptides/proteins being identified, most probablydue to a better dissolution of the proteins beforedigestion.

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Spectrum Mill MS proteomics workbench enabledcomparison of individual samples or even multiplesamples/experiments through its protein-proteinsummary report (Figure 4). With this report, allassigned peptides from different samples/experi-ments are merged together to identify the proteinsin a first step. The individual peptides from eachsample/experiment are then displayed in separatecolumns, together with the protein sequence cov-erage and number of identified peptides from allpeptides.

A 1D nano-LC/MS analysis of a 10 mm2 post-mortem tissue sample resulted in the positiveidentification of 26 proteins from a single sample

Agilent Technologies Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

(Figures 5 and 6). Due to the large number of digested peptides from the tissue sample, the MS base peak chromatogram (BPC) showed hundreds of peaks. The MS/MS total ion chromatogram(TIC) showed intensities comparable to the BPC intensities, since the fragmentation efficiency of peptides in the XCT ion trap is typically close to 100%. More than 150 MS/MS spectra were auto-matically assigned to peptide sequences and 26 protein hits were created with absolute scores greater than 15. Future work will include acquiring more in-depth protein profiles of tissue samples by working with larger micro-dissected areas, combined with multi-dimensional LC sepa-ration techniques.

Figure 4. Comparative study of different digestion protocols; left column (Sample 5) represents proteins identified using the TFEprotocol, right column (Sample 4) represents proteins identified using the urea protocol. Color coding indicates relative proteinabundance, with white indicating not detected and progressively darker colors indicating progressively higher abundance.

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Agilent Technologies Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

Conclusions

Tissue samples prepared with LMPC enable directaccess to soluble proteins for further investiga-tion. A digestion protocol in mixed organic sol-vents was developed, leading to better sequencecoverage and protein identification by LC/MS.One-dimensional nano-LC/MS/MS ion trap massspectrometry using a high capacity ion trap gener-ated positive identification of up to 30 proteinsfrom tissue areas as small as 5 mm2.

Figure 5. Base Peak Chromatogram (MS) and Total Ion Chromatogram (MS/MS) of a 10 mm2 tissue sample

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050-1001.D: BPC 100-2200 ±All MS

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Agilent Technologies Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS

Figure 6. SpectrumMill results from a 10 mm2 post-mortembrain tissue sample

Acknowledgments

The authors thank Jose Meza, Steven Fischer, andChristine Miller of Agilent Technologies who con-tributed the TFE/trypsin digestion protocol forwhich publication is pending.

Authors

Bernd Sägmüller is an Application Specialist at PALM Microlaser Technologies. Hans A.Kretschmar is the Chairman of the Institute ofNeuropathology and Manuela Neumann is a med-ical doctor, MD and Neuropathologist at Ludwig-Maximilians-Universität München. ChristianSauber is an Applications Chemist at AgilentTechnologies.

www.agilent.com/chem

© Agilent Technologies, Inc. 2004

For Research Use only. Not for use in diagnostic procedures.

Information is subject to change without notice.

Printed in the U.S.A. May 31, 20045989-0895EN

Agilent Technologies Identification of Proteins in Post-Mortem Human Brain Tissue by LaserMicrodissection/Pressure Catapult and Nano-LC/MS/MS