Patent Application
20070069122
This application claims the benefit of European Application Nos. 05104829.6, filed Jun. 3, 2005 and 06111461.7, filed Mar. 21, 2006, both of which are hereby incorporated by reference in their entirety. This application also claims the benefit of co-filed US Application No. 23145, entitled “Transthyretin as a Marker for Nephrotoxicity”, which is likewise incorporated by reference in its entirety.
The present invention relates to methods for de novo in situ protein biomarker identification, and more particularly, methods for de novo in situ protein identification (protein biomarker) wherein proteins are directly eluted from the tissue region of interest (without any need for tissue homogenization).
Proteomics techniques have the potential to correlate differential protein expression with environmental disease or toxicant exposure. High resolution two-dimensional gel electrophoresis technology in combination with mass spectrometry have demonstrated the usefulness of proteomics approaches especially in the toxicology area (Bandara, L. R., and Kennedy, S. (2002) Drug Discovery Today 7, 411-418). Of particular interest is the early detection of disease but also the rapid screening against experimental compounds either for toxicity or efficacy (Petricoin, E. F., Rajapaske, V., Herman, E., Arekani, A. M., Ross, S., Johann, D., Knapton, A., Zhang, J., Hitt, B. A., Conrads, T. P., Veenstra, T. D., Liotta, L. A., and Sistare, F. D. (2004) Toxicologic Pathology 32, 122-130). The feasibility of toxicoproteomics studies has been demonstrated both for the identification of predictive markers and for the elucidation of toxicant mode (Witzmann, F. A., and Li, J. (2004) Proteomics in Nephrology 141, 104-123; Bandara, L. R., Kelly, M. D., Lock, E. A., and Kennedy, S. (2003) Toxicological Sciences 73, 195-206).
MALDI imaging mass spectrometry (IMS) represents a new technique for the direct peptide and protein analysis from a tissue thin section by conventional MALDI-TOF MS (need definition) instruments (Chaurand. P, Schwartz, S. A, Billheimer, D., Xu, B. J., Crecelius, A. Caprioli, R. M. (2004) Anal. Chem. 76, 1145-1155). The generated two-dimensional ion density map can then be used to derive the relative abundance and spatial distribution of proteins in situ. The usefulness of this technique has been demonstrated in the biomarker field in experimental models (Pierson, J., Norris, J. L., Aerni, H.-R., Svenningsson, P., Caprioli, R. M., and Andrén, P. E. (2004) Journal of Proteome Research 3, 289-295, Chaurand, P., DaGue, B. B., Pearsall, R. S., Threadgill, D. W., and Caprioli, R. M. (2001) Proteomics 1, 1320-1326) and in clinical settings, for instance for accurate and sensitive classification of non-small-cell lung cancer tumor biopsies (Yanagisawa, K., Shyr, Y., Xu, B. J., Massion, P. P., Larsen, P. H., White, B. C., Roberts, J. R., Edgerton, M., Gonzales, A., Nadaf, S., Moore, J. H., Caprioli, R. M., and Carbone, D. P. (2003) The Lancet 362, 433-439).
Currently known methods for identification of de novo protein in diseased tissues (ie biomarkers) by MALDI MS (need definition) involves tissue homogenization followed by one- or two dimensional electrophoresis and identification of modulated protein levels by MALDI MS. However, the need for tissue homogenization in these methods present problems with regard to increased time to prepare the homogenization, contaminant issues relating from said homogenization etc. A method for identification without tissue homogenization would improve sensitivity in keeping the spatial resolution of the tissue (i.e. less contaminants), and would not need mixing or lysing steps (i.e. simpler to perform).
According to the present invention a method is provided for in situ identification of a protein biomarkers in MALDI IMS. Specifically, the present invention provides a method to identify a polypeptide which is accumulated or depleted in situ in a specific tissue without tissue homogenization, comprising the steps of
In a preferred embodiment of the method of the present invention, a tissue thin section from a specific tissue of interest is provided. Preferably, said tissue thin section is 5 to 15 μm thick. The tissue section can be further processed, e.g. by fixation with an alcohol, acetonitrile etc. Preferably, the fixative is ethanol. Then, a MALDI matrix is deposited on said tissue section.
Preferably, the MS spectra is obtained by laser beam. Preferably the method does not require tissue homogenization.
The term “MALDI” as used herein refers to Matrix-Assisted Laser Desorption Ionization.
The term “MALFI-TOF MS” as used herein refers to Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry.
The term “MALDI MS” as used herein refers to Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.
The term “MALDI IMS” as used herein refers to Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry.
The term “Tandem MS” as used herein refers to Tandem Mass Spectrometry.
The term “MALDI matrix” as used herein refers to Matrix-Assisted Laser Desorption Ionization matrix.
All patents, patent applications and publications herein, both supra and infra, are hereby incorporated by reference.
According to the present invention a method is provided for de novo in situ identification of protein biomarkers by the novel combination of MALDI IMS with associated techniques, more specifically, the present invention provides a method to identify a polypeptide which is accumulated or depleted in situ in a specific tissue, comprising the steps of
The invention also provides a method for in situ identification of a protein biomarkers in MALDI IMS. Specifically, the present invention provides a method to identify a polypeptide which is accumulated or depleted in situ in a specific tissue, comprising the steps of
In a preferred embodiment of the method of the present invention, a tissue thin section from a specific tissue of interest is provided. Preferably, said tissue thin section is 5 to 15 μm thick. The tissue section can be further processed, e.g. by fixation with an alcohol, acetonitrile etc. Preferably, the fixative is ethanol. Then, a MALDI matrix is deposited on said tissue section. Preferably, the MS spectra is obtained by laser beam. The method of the present invention avoids the need for tissue homogenization while improving sensitivity in keeping the spatial resolution of the tissue (less contaminants) and is simpler to perform (no need for mixes, lysis etc.).
Said tissue section can originate from any tissue of interest of any multicellular organism. For example, such tissue can originate from a human subject, any non-human multicellular organism, including non-human mammal, e.g. mouse, rat, rabbit, swine, non-human primate, but also from other organisms such as e.g. insects, nematodes or plants.
MALDI imaging mass spectrometry (IMS) represents a new technique for the direct peptide and protein analysis from a tissue thin section by conventional MALDI-TOF MS instruments (Chaurand. P, Schwartz, S. A, Billheimer, D., Xu, B. J., Crecelius, A. Caprioli, R. M. (2004) Anal. Chem. 76, 1145-1155). The generated two-dimensional ion density map can then be used to derive the relative abundance and spatial distribution of proteins in situ. The usefulness of this technique has been demonstrated in the biomarker field in experimental models (Pierson, J., Norris, J. L., Aerni, H.-R., Svenningsson, P., Caprioli, R. M., and Andrén, P. E. (2004) Journal of Proteome Research 3, 289-295, Chaurand, P., DaGue, B. B., Pearsall, R. S., Threadgill, D. W., and Caprioli, R. M. (2001) Proteomics 1, 1320-1326) and in clinical settings, for instance for accurate and sensitive classification of non-small-cell lung cancer tumor biopsies (Yanagisawa, K., Shyr, Y., Xu, B. J., Massion, P. P., Larsen, P. H., White, B. C., Roberts, J. R., Edgerton, M., Gonzales, A., Nadaf, S., Moore, J. H., Caprioli, R. M., and Carbone, D. P. (2003) The Lancet 362, 433-439).
Suitable MALDI matrices are well known in the art (see e.g. Trauger et al., Spectroscopy 2002, 16(1), 15-28). Some MALDI matrices are more suitable for peptide analysis (e.g. alpha cyano cinnamic acid), but they are not optimal for protein applications. Preferably, said MALDI matrix is a matrix suitable for protein applications, such as sinnapinic acid as a non limiting example.
In a further step, MALDI MS spectra are acquired by exposing the tissue section to a laser beam. Laser beams suitable to generate MALDI MS spectra are well known in the art. As a non limiting example, a standard 337-nm N2 laser operating at 50 Hz can be used. Preferably, said laser has a laser spot size of about 10 to about 100, more preferably of about 40 to about 60, most preferably of about 50 μm. A too small (less than about 10 μm) laser spot size will not produce any signal, wherein a too large (more than about 100 μm) laser spot size will present problems with mass accuracy readings.
The MALDI MS spectra generated above are then analyzed. Tools for analysis of MALDI MS spectra are well known to the skilled person and can be obtained commercially. One such tool is e.g. the ProTS-Data software from Efeckta Technologies Inc. Peaks of interest are then selected. Such selection may, as a non-limiting example, be based on a ranking of spectral features according to the extent of observed differences, e.g. using the Weighted Flexible Compound Covariate Method (WFCCM) (Shyr, Y., and KyungMann, K. (2003) A Practical Approach to Microarray Data Analysis, ed D. Berrar).
In a further step of the method of the present invention, proteins are eluted from a tissue sample corresponding to the tissue thin section hereinbefore described. Said tissue sample may be a large piece of tissue or a microdissected tissue. Preferably, said tissue sample is a tissue thin section corresponding to the tissue thin section used for MALDI MS analysis hereinbefore described. Said tissue thin section can also be the same tissue thin section as the one used for MALDI MS in the previous steps. The elution of proteins is achieved by protein micro-extraction, which can be achieved, as a non-limiting example, by directly pipetting up and down a small volume of extraction solvent on the region of interest of the tissue section. This process can be repeated several times and samples be pooled to obtain enough material for subsequent protein identification.
In a next step, the eluates are fractionated. Fractionation can be performed using HPLC or other liquid chromatographic methods known to one of ordinary skill in the art.
The fraction containing the mass (peak) of interest is identified by MALDI TOF MS, or any other spectrometric method known to one of ordinary skill in the art. Preferably, the fraction containing the mass (peak) of interest is identified by MALDI TOF MS.
The protein contained in said fraction is then identified by performing tandem mass spectrometry and analyzing the resulting spectrum. As a non-limiting example, the protein can be identified with the Mascot MS/MS ion search program (MatrixScience) using the SwissProt database. Other program(s) and/or database(s) may be used as needed/desired and as known to one of ordinary skill in the art.
The accumulation of the protein transthyretin identified in the kidney cortex in nephrotoxicity according to the example provided below is a processed protein which accumulates in a tissue where it is not intrinsically expressed, therefore, it would not be possible to identify said biomarker by methods based on DNA expression.
Permission for animal studies was obtained from the local regulatory agencies, and all study protocols were in compliance with animal welfare guidelines. Male HanBrl: Wistar rats of approximately 12 weeks of age (300g±20%) were obtained from BRL, Füllinsdorf, Switzerland. The animals were housed individually in Macrolone cages with wood shavings as bedding at 200C and 50% relative humidity in a 12-hr light/dark rhythm with free access to water and Kliba 3433 rodent pellets (Provimi Kliba AG, Kaiseraugst, Switzerland).
Animals were treated for 7 consecutive days with 100 mg/kg/day Gentamicin (dissolved in saline) or vehicle by subcutaneous (sc) injections and sacrificed 24 hours after the last application by CO2 inhalation. Immediately preceding sacrifice, terminal blood samples for clinical chemistry investigations were collected from the retroorbital sinus under isofluoran anesthesia.
Representative kidney samples were fixed in 10% neutral buffered formalin. All samples were processed using routine procedures and embedded in Paraplast. Tissue sections approximately 2-3 microns were cut and stained with hematoxylin-eosin (HE) or periodic acid-Schiff (PAS).
The differential protein expression was studied within the areas of the kidney (Cortex, Medulla, Papilla) to investigate whether the histopathologically-observed toxicity of gentamicin could be correlated by IMS. Several protein peaks in the cortex area were significantly modulated upon gentamicin administration.
Marked degeneration/regeneration of proximal tubules (grade 4) was clearly observed in rats treated with 100 mg/kg/day gentamicin for 7 days. This finding was correlated to the analysis of rat kidney thin sections by MALDI IMS. 9 control and 9 gentamicin-treated rat kidneys thin sections (3 animals, 3 sections from each animal; the thin section was spotted with matrix as shown in
Kidneys were dissected from rats, snap frozen on dry ice and stored at −80° C. prior to further processing. 12 μm sections were obtained on a cryostat (LEICA CM 3000, Leica Microsystems) at −18° C. and deposited on ITO-coated conductive glass slides (Indium Tin Oxide 50×75×0.9 mm, Delta Technologies). Sample preparation were performed according to published procedure (Schwartz, S. A., Reyzer, M. L., and Caprioli, R. M. (2003) Journal of Mass Spectrometry 38, 699-708). Tissue sections were then fixed by immersion in ethanol bathes and let to dry 30 minutes under vacuum. During all the process the sections were stored as much as possible under vacuum. Deposition of MALDI matrix was done manually with a pipette. 200 nL of freshly prepared sinapinic acid solution (3,5-dimethoxy-4-hydroxycinnaminic acid, Fluka, 20 mg/ml in water/acetonitrile 1:1, 0.1% trifluoroacetic acid) was deposited onto the tissue. After crystallization, an additional 200 nL was layered onto the spots and the matrix was let to dry at room temperature. The sample was then kept under vacuum if not analyzed immediately by mass spectrometry.
MALDI MS spectra were acquired using an UltraFlex MALDI ToF/ToF instrument (Bruker Daltonics) with a standard 337-nm N2 laser operating at 50Hz and a laser spot size of 50 μm. The instrument was operated in positive linear mode (2 kDa-30 kDa) at constant laser power. A total of 8 times 100 laser shots were averaged for each manually deposited matrix droplets. Spectra were externally calibrated using a protein calibration standard solution (Insulin, Ubiquitin I, Cytochrom C, Myoglobin; Bruker Daltonics). Internal calibration was performed post-acquisition using redundant known peaks from the spectra (Hemoglobin α-chain, Thymosin β-4, Cytochrome c oxidase polypeptide VIIa L, Ubiquitin, Cytochrome c oxidase polypeptide VIb).
The spectra were processed using a combination of tools available commercially and developed in-house at Vanderbilt University. Baseline subtraction, normalization, peak detection, and spectral alignment were performed using the ProTS-Data software (Efeckta Technologies, Inc.) All spectra were baseline corrected to remove contributions of chemical noise to the signal. This step was performed by locally estimating the baseline and subtracting the spectrum from the raw data. For the present data set the software was set to consider a window width 10 times that of the peak width to account for the differences in resolution for different m/z regions. The baseline corrected spectra were normalized in intensity by scaling the spectra to a common total ion current. Peak detection was performed using ProTS-Data and a list of 13 common peaks was selected to be internal alignment points using a quadratic calibration function. The criterion used to select common peaks were that the peak must occur in greater than 80% of the spectra to be aligned, must not have interfering or overlapping peaks, and the peaks must have a standard deviation of the observed centroid value less that 7 mass units. To avoid error caused by extrapolation during the alignment step, alignment peaks were chosen across the entire mass range of interest ranging from 2500 to 15000 mass units. The peak lists containing the centroid value and normalized intensity are exported to individual files.
The peak lists were binned according to their m/z value using an in-house program developed at Vanderbilt University. The exported MALDI-TOF MS peaks were aligned across samples by use of a genetic algorithm parallel search strategy (Yanagisawa, K., Shyr, Y., Xu, B. J., Massion, P. P., Larsen, P. H., White, B. C., Roberts, J. R., Edgerton, M., Gonzales, A., Nadaf, S., Moore, J. H., Caprioli, R. M., and Carbone, D. P. (2003) The Lancet 362, 433-439). Briefly, peaks were binned together such that the number of peaks in a bin from different samples is maximized while the number of peaks in a bin from the same sample is minimized. A series of mass windows or peak bins were generated that separated similar peaks across multiple spectra. The spectral features were ranked according to the extent of the observed difference in order to determine relevant biomarkers for gentamicin induced kidney toxicity using the Weighted Flexible Compound Covariate Method (WFCCM). This strategy, described by Shyr et al.(Shyr, Y., and KyungMann, K. (2003) A Practical Approach to Microarray Data Analysis, ed D. Berrar), uses a combination of 6 different statistical tests to compute a ranking based on the difference observed between treated and control.
A simple data cluster analysis was successful in differentiating the control from the treated conditions. As expected, most of the peaks discriminating the control from the treated were found in the cortex. Table 1 shows the proteins that were statistically identified as being up or down regulated in the control versus the treated samples in the cortex.
Ranking by:
1) WMA
2) Maximum average S/N
3) p-value
In this dataset, a peak at mass m/z 12 959 was ranked in 1st position in the cortex with an average signal-over-noise of 5 in gentamicin-treated rat kidney and completely absent in the controls (
A liquid micro-extraction was performed in the cortex area.
Protein micro-extraction was performed by directly pipetting up and down for a few seconds 1 μl of solvent (water/acetonitrile 1:1, 0.1% trifluoroacetic acid) on the cortex area of the kidney. This process was repeated several times on several samples to obtain a pooled sample volume of approximately 20 μl. The sample was then dried in a speed vac and resolubilized in water/acetonitrile 95:5, 0.1% trifluoroacetic acid.
The resulting protein mixture was fractionated by reverse-phase liquid chromatography.
Protein fractionation was performed using a standard Agilent 1100 series HPLC (Agilent Technologies) equipped with a 1 mm i.d. polymeric column (219TP51 10, Vydac) operated at 50 μl/min. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in acetonitrile. Proteins were eluted using the following program: the gradient was started after 5 minutes with 5% B up to 35% in 7 minutes, then to 60% B in 34 minutes, and then increased to 90% B in 0.5 minute and hold for 10 minutes. Fractions were collected every 30 sec by time immediately after the beginning of the run up to 48 minutes directly in a 96 well microtiterplate (Greiner bio-one). If necessary, a sample was fractionated over several HPLC runs and corresponding fractions were then combined for analysis.
The obtained LC fractions were surveyed by MALDI-TOF MS by spotting 1 μl of the fraction with 1 μl of 1 g/L sinapinic acid solution in water/acetonitrile 90:10, 0.1% TFA on a 384 MALDI Anchor target (Bruker Daltonics).
The presence of the peaks of interest was assessed by comparing the IMS-generated spectra with the LC-fractionated spectra.
The fraction containing the protein species of interest was then further analyzed by nano-ESI mass spectrometry and the proteins of interest were identified by tandem mass spectrometry.
For protein identification, the HPLC fraction containing the mass of interest was dried in a speed vac and resolubilized in water/acetonitrile 1:1 containing 1% formic acid for identification using an Applied Biosystems Q-Star Pulsar operated under standard operating conditions in nano-electrospray mode. One of the multiply charged species of the mass of interest was selected for tandem mass spectrometric analysis, its tandem mass spectrum was recorded and manually interpreted. Protein identification was performed with the Mascot MS/MS ion search program (Matrix Science, http://www.matrixscience.com) using the SwissProt database (release 46.6) and a tolerance of 1.0 Da for fragment and precursor masses.
The strategy that was followed to identify this peak is illustrated in
One of the biomarkers modulated upon gentamicin administration was identified as transthyretin (prealbumin), a 13 kDa blood transporter protein. This finding was confirmed by Western blot using an antibody highly specific for this protein showing that this protein was significantly more abundant in kidneys from gentamicin-treated rats than in control rats (
Tissue extracts from control and treated rat kidneys were obtained by homogenization of a piece of cortex in a 10 mM Tris-HCl buffer pH 7.6 containing 250 mM sucrose and protease inhibitors (Roche Complete). The tissue extract was subjected to three successive centrifugation steps at 4° C. (10 min at 680 g; 10 min at 10 000 g; 1 h at 100 000 g) whereas the pellets were discarded and the supernatant of the last centrifugation step was kept as the kidney cytosolic fraction.
1 μl of the kidney cytosolic fraction and of the relevant HPLC fraction were subjected to electrophoresis using a 4-20% Tris-Glycine SDS-PAGE gel (Invitrogen) and the proteins were subsequently transferred to a PVDF membrane. Blots were blocked in 5% milk in PBS containing 0.1% Tween 20 for 1 h and incubated overnight at room temperature with an anti-sheep prealbumin antibody (Abcam) in PBS containing 5% milk and 0.1% Tween 20. After washing with PBS containing 0.1% Tween 20, blots were incubated for 1 h with horseradish peroxidase conjugated with an anti-sheep IgG antibody (Silenus Laboratories). Antibody binding was detected using ECL western blotting reagents (Amersham).
Similarly, the m/y species 12959, which was identified by tandem mass spectrometry as transthyretin S28-Q146, was only present in the mass spectrum of the gentamicin-treated sample in situ and in the HPLC fraction D10.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05104829.6 | Jun 2005 | EP | regional |
| 06111461.7 | Mar 2006 | EP | regional |
