- Email: [email protected]
Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections
676
Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections Pierre Chaurand, Sarah A Schwartz and Richard M Caprioli* MALDI MS imaging mass spectrometry can be used to map the distribution of targeted compounds in tissue sections with a spatial resolution currently of about 50 µm, providing important molecular information in many areas of biological research. After matrix application, a raster of a section by the laser beam yields ions from compounds in a tissue mass-tocharge range from 1000 to over 100 000. Two-dimensional intensity maps can then be reconstructed to provide specific molecular images of a tissue. Addresses Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-6400, USA *e-mail: [email protected] Current Opinion in Chemical Biology 2002, 6:676–681 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 August 2002 Abbreviations MALDI matrix-assisted laser desorption ionization TOF time-of-flight
Introduction Imaging mass spectrometry can be used to measure the spatial arrangement and relative concentration of compounds in biological samples, from elements to large proteins [1•]. One recently developed imaging technology utilizes matrix-assisted laser desorption ionization (MALDI) and time-of-flight mass spectrometry (TOF MS) to generate profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections [1•,2••]. This allows specific information to be obtained on the local proteomic composition, relative abundance and spatial distribution of these components. Results from such imaging experiments yield a wealth of information, allowing investigators to measure and compare many of the major molecular components of the section to gain a better understanding of biological processes involved. MALDI mass spectrometry was first described in the late 1980s [3,4]. The sample preparation procedure involves mixing of a solution of a low molecular weight matrix molecule with analyte molecules (i.e. peptides and proteins) on the target plate. Evaporation of the solvent results in the formation of matrix-analyte co-crystals. Irradiation of these crystals by a short duration laser pulse initiates the desorption and ionization events, leading to the formation of singly charged protonated analyte molecular ions ([M+H]+). Recent improvements in time-of-flight technology [5,6] have allowed MALDI MS to become an extremely
accurate and sensitive tool for the detection, identification and characterization of peptides and proteins [7–9,10•,11,12,13•]. Proteins having molecular weights above 100 kDa can be detected for sample amounts in the femtomole range. Protein identification is accomplished from peptide maps following trypsin digestion and matching the results with theoretical digests of proteins in a database. For still higher confidence identification, the sequence analysis of one or more peptide fragments is obtained by tandem mass spectrometry, followed by sequence matching in the protein database [14–16].
Profiling proteins from blots of fresh tissue sections Over the past few years, several investigators have optimized protocols to generate protein profiles by MALDI MS from cells, groups of cells and small tissue sections immobilized on a target plate [17–26,27••]. Using MALDI MS, we have measured proteins in fresh tissues after transfer by contact blotting of the tissue on an active surface such as C18 [28] or a carbon-filled polyethylene membrane [29]. The latter is especially useful because it has both electrical conductivity and protein adhesion properties. Matrix was deposited on the blotted areas and, upon analysis, reproducible tissue-specific protein profiles were generated for a molecular range exceeding 100 kDa. Figure 1 presents the protein profiles obtained by contact blotting the different lobes of a mouse prostate on a polyethylene membrane. Contact blotting was performed for 5 min, covering the tissue samples with a glass slide to avoid dehydration. The blotted areas were rinsed with water to remove cell debris and tissue fragments as well as excess physiological salts and blood before matrix deposition. Data acquisition was performed in the linear mode under optimized delayed extraction conditions [5,6] on an Applied Biosystems DE-STR MALDI TOF mass spectrometer. After analysis of the different lobes of the prostate gland, very different and specific profiles containing over 300 individual signals, some at low intensities, were recorded in a m/z range up to 100 000. For example, the group of signals between m/z 21 800 and m/z 24 800, identified as different glycoforms of spermine-binding protein, have been found expressed at a much higher level in the ventral prostate lobe [30]. Similarly, the signal detected at m/z 75 094 has been detected at much higher intensity in the anterior prostate lobe. The profiles obtained after tissue blotting are extremely reproducible from animal to animal in the same strain line [29]. Using this new protein profiling approach, we have studied proteins present in sections of colon and prostate cancers, identifying several tumor-specific proteins [31,32].
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
677
Figure 1 Protein profiles obtained by MALDI MS after fresh tissue blotting on a polyethylene membrane for the mouse anterior (AP), dorsal (DP), lateral (LP) and ventral prostate (VP) lobes. α+ and β+ are α and β hemoglobin, respectively.
α+ β+ AP
DP
– –
LP
VP
6800
11 600
16 400
24 800
21 800
21 200
AP
75 094
Relative intensities
– –
DP
LP
VP 26 000
36 800
47 600
58 400
69 200
80 000
Mass (m/z) Current Opinion in Chemical Biology
Profiling and imaging of proteins directly from thin tissue sections Direct analysis of a thin tissue section by MALDI imaging mass spectrometry produces an array of signals in a mass range from 2 kDa to above 100 kDa, corresponding to hundreds of peptides and proteins present in the tissue. Figure 2 presents the protein profile obtained from a 12 µm human brain tissue section after application of sinapinic acid as matrix (saturated solution in a solution of 50/50/0.1 H2O/acetonitrile/trifluoroacetic acid, in volume). Thin sections, typically 12 µm, were cut at –15°C from snap frozen tissue samples using a cryostat. The sections were thaw-mounted on a target plate, allowed to dry in a dessicator for one hour and spotted with 200 nl of matrix before mass spectrometric analysis. In Figure 2, in the insert region alone in the m/z range from 5400 to 8400, over 60 individual mass signals were detected.
Principle of imaging mass spectrometry MALDI imaging mass spectrometry can be used to map the distribution of peptides and proteins directly from thin tissue
sections. Matrix is deposited uniformly over the section, and a raster of the tissue is performed over a predetermined twodimensional array or grid, generating a full mass spectrum at each grid coordinate. With commercially available instruments, the laser spot size on target can be reduced to about 50 µm in diameter after some fine-tuning of the optics, defining the actual imaging resolution. Each spectrum is the result of the average of 15 to 50 or more consecutive laser shots at each coordinate. From the intensity of a given m/z value monitored in each spectrum, a density map or image can be constructed. Virtually all of the signals detected from the section can be used to generate specific molecular weight images from a single raster of the tissue. The concept of MALDI-MS-based imaging mass spectrometry was introduced in 1997 by Caprioli and co-workers [28] to image fresh tissue slices either after coating the sample with matrix or after blotting the tissue slice on a target coated with C18 beads. In the latter case, a thin coat of matrix was deposited on the blotted area after removal of the tissue. Molecular ion
678
Analytical techniques
Figure 2
6086
7866
6971 5767 6664 6705
0 2000
7600
18 521
22 772
18 800
24 909
17 255
13 200
7800
7200
20 926
20
7383
6600
12 347
x3
6000
13 786 15 124 15 868
5358
5172
5400
40
7662
9956 10 625
8566
60
4039
% Intensity
80
Protein profile obtained by MALDI MS after matrix deposition (spotted with 200 nl of matrix) on a 12 µm human brain tissue section. Over 300 individual mass signals, some with low intensities, were detected.
7754
24 400
27 795
5651
4965
100
30 000
Mass (m/z) Current Opinion in Chemical Biology
images were successfully generated for rat splenic pancreas and for an area of the rat pituitary where over 50 different peptides were observed as well as some of their precursors, isoforms and metabolic fragments. Similarly, Garden and Sweedler [33] have mapped the intensity of standard peptides in MALDI samples prepared in a single drop of sample. Stoeckli et al. [34] developed a new imaging computer algorithm allowing both instrument control and data acquisition and processing for MALDI MS imaging of thin tissue sections. Using this approach, an image of a thin section of a human glioblastoma xenograph was obtained [2••].
Sample preparation and handling Careful sample preparation and handling is important to obtain high-quality images. For best results for high-resolution imaging, matrix must be deposited on the surface of the tissue in a homogeneous coat. Three conditions should be maintained to the greatest extent possible. First, the deposition process must not disperse or translocate proteins within the section. Second, the matrix solution must wet the tissue surface to form crystals, which contain co-crystallized proteins. Third, the crystal dimensions must be smaller than the image resolution. The following procedure is typical of sample preparation and can be used with most tissues. Immediately following
dissection, the tissue sections are loosely wrapped in aluminum foil and plunged into liquid nitrogen. Frozen tissue should be stored at –80°C until analysed. Twelvemicron sections are cut with a cryostat. This thickness is not critical and was chosen because of easy handling. The sample coating method that has proven to be the most successful thus far consists of air spraying matrix on the tissue. Matrix (saturated solution in a solution of 50/50/0.1 H2O/acetonitrile/ trifluoroacetic acid, in volume) is sprayed over the section using a commercially available glass spray nebulizer (similar to those used to coat thin-layer chromatography slides) connected to a nitrogen bottle (nebulizing gas) to minimize contamination. The coating procedure is as follows. About 500 µl of matrix are sprayed on the section at a distance of about 20 to 30 cm and the sample allowed to dry at room temperature (only a small fraction of the total volume is actually deposited on the section). It is important that the combination of the spray deposit and spray distance be optimized to avoid excessive wetting of the section, inevitably leading to protein delocalization. This spray cycle is repeated up to 10 times, allowing some air drying between cycles (30 s to 1 min) to generate a homogeneous crystal field. Sinapinic acid as matrix in the indicated solvent system typically yields crystals ranging in size from 5 µm to 25 µm in length. The quality of the mass
Figure 3 legend MALDI Imaging of a transversal rat brain section. (a) Survey profile with data acquisition taken randomly across the section. (b) Optical image of the section before matrix application. The general outlines of
the section as well as several features visible in the section have been delineated. (c)–(g) Ion density maps obtained at different m/z values. The section was imaged with a resolution of 180 µm.
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
679
Figure 3
(a) 100
7059
7532
6844, (d)
80 6714 6541
6909 7275
6062
5890 5632, (c)
% Intensity
60
6882
14117
40
7059 11 338, (e) 11 297
12 122 15 184
20
9969 15 807
3767 4960
18 393, (f) 21 882, (g)
0 2000
6600
11 200
15 800
20 400
25 000
Mass (m/z)
4 mm
(b) Optical image
(c) m/z 5632
optical
image
0
100%
a)
(d) m/z 6844
(e) m/z 11 338
(f) m/z 18 393
(g) m/z 22 920 Current Opinion in Chemical Biology
680
Analytical techniques
spectrometric data generated from this spray procedure is essentially the same as that generated using the drop deposition method.
spectrometry may also one day permit assessment of surgical margins at the cellular and molecular level.
Acknowledgements Application: imaging the rat brain The unique symmetry and well-characterized anatomy of brain allow it to be an excellent tissue model. Transversal rat brain sections were cut, thaw-mounted on the target plate (Figure 3b) and coated with matrix. First, a survey scan was performed with data acquisition taken randomly across the section to generate an average protein profile from this section (Figure 3a). Over 200 individual mass peaks were detected in a m/z range up to 40 000. Figure 3b presents an optical image of the brain section before matrix deposition. The general outlines of the section as well as several features visible in the section have been delineated. The section was scanned by acquiring 74 × 75 points with a resolution of 180 µm by averaging spectra produced by 15 laser shots using the automated imaging computer algorithm [34]. In this scan, the intensity of 45 different mass signals was monitored. Figure 3c–g presents five ion density maps obtained for different protein signals; some of these, in particular m/z 6844, have low intensities (see signals c–g in Figure 3b). As expected, some proteins were found to be very specific for a given brain region. This is particularly striking for the density maps of the proteins detected at m/z 5632 and m/z 18 393, which are almost ‘negatives’ of each other.
Conclusions Imaging mass spectrometry is still in an early stage and many developments and improvements in both sample preparation and handling as well as instrumentation are to be expected within the coming years. Items such as imaging acquisition speed, image resolution and data mining tools need to be further developed. Nonetheless, the potential of such a molecular imaging tool is enormous. The fundamental contributions of the technology in rapidly providing molecularweight-specific maps or images, at relatively high resolution and sensitivity, will provide a powerful tool for the investigation of cellular processes in both health and disease. Imaging mass spectrometry is of extraordinary benefit as a discovery tool where a tissue section can be investigated without necessarily knowing in advance what specific proteins have changed in a comparative study. Profile and image comparison between tissues allows researchers to easily identify differences in protein expression. Such experiments have already been performed comparing the profiles obtained for healthy and cancerous mouse colon tissue samples and, from these data, several cancer biomarkers were identified [31]. This study also provided valuable information on the relative concentration of proteins within the section. For clinicians, this will permit molecular assessment of tumor biopsies with the potential to identify sub-populations that are not evident based on the cellular phenotype determined microscopically. Further, it is not inconceivable that imaging mass
The authors thank Robert Weil (Department of Neurosurgery, Vanderbilt University Medical Center) for providing the human brain tissue section and Ariel Deutch (Departments of Psychiatry and Pharmacology, Vanderbilt University Medical Center) for providing the rat brain tissue sample. The authors also acknowledge funding by the National Institutes of Health (grant GM 58008).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1. •
Todd PJ, Schhaaff TG, Chaurand P, Caprioli RM: Organic ion imaging of biological tissue with SIMS and MALDI. J Mass Spectrom 2001, 36:355-369. Reference article describing the principle of imaging mass spectrometry and introducing its potential in cancer research. 2. ••
Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493-496. This tutorial article reviews the different techniques and methodologies employed for imaging organic compounds from thin tissue sections by mass spectrometry. 3.
Karas MH, Hillenkamp F: Laser desorption ionization of proteins with molecular masses exceeding 10000 Daltons. Anal Chem 1988, 60:2299-2301.
4.
Hillenkamp F, Karas M, Beavis RC, Chait BT: Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal Chem 1991, 63:1193A-1202A.
5.
Brown RS, Lennon JJ: Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laserdesorption ionization linear time-of-flight mass-spectrometer. Anal Chem 1995, 67:1998-2003.
6.
Vestal ML, Juhasz P, Martin SA: Delayed extraction matrix-assisted laser-desorption time-of-flight mass-spectrometry. Rapid Commun Mass Spectrom 1995, 9:1044-1050.
7.
Roepstorff P: Mass spectrometry in protein studies from genome to function. Curr Opin Biotechnol 1997, 8:6-13.
8.
Russell DH, Edmondson RD: High-resolution mass spectrometry and accurate mass measurements with emphasis on the characterization of peptides and proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Mass Spectrom 1997, 32:263-276.
9.
Lahm HW, Langen H: Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis 2000, 21:2105-2114.
10. Pandey A, Mann M: Proteomics to study genes and genomes. • Nature 2000, 405:837-846. This review article presents a clear and comprehensive overview of the role of mass spectrometry in modern proteomics. 11. McDonald WH, Yates JR: Proteomic tools for cell biology. Traffic 2000, 1:747-754. 12. Aebersold R, Goodlett DR: Mass spectrometry in proteomics. Chem Rev 2001, 101:269-295. 13. Godovac-Zimmermann J, Brown LR: Perspectives for mass • spectrometry and functional proteomics. Mass Spectrom Rev 2001, 20:1-57. Very detailed review article on the potential of mass spectrometry for the detection, identification and further characterization of peptides and proteins. 14. Mann M, Wilm M: Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 1994, 66:4390-4399. 15. Yates JR: Database searching using mass spectrometry data. Electrophoresis 1998, 19:893-900.
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
16. Borchers C, Peter JF, Hall MC, Kunkel TA, Tomer KB: Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. Anal Chem 2000, 72:1163-1168. 17.
Gusev AI, Vasseur OJ, Proctor A, Sharkey AG, Hercules MH: Imaging of thin-layer chromatograms using matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 1995, 67:4565-4570.
18. Dreisewerd K, Kingston R, Geraerts WPM, Li KW: Direct mass spectrometric peptide profiling and sequencing of nervous tissues to identify peptides involved in male copulatory behavior in Lymnaea stagnalis. Int J Mass Spectrom 1997, 169:291-299. 19. Redeker V, Toullec JY, Vinh J, Rossier J, Soyez D: Combination of peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunodetection on single glands or cells. Anal Chem 1998, 70:1805-1811. 20. Jimenez CR, Burlingame AL: Ultramicroanalysis of peptide profiles in biological samples using MALDI mass spectrometry. Exp Nephrol 1998, 6:421-428. 21. Jimenez CR, Li KW, Dreisewerd K, Spijker S, Kingston R, Bateman RH, Burlingame AL, Smit AB, van Minnen J, Geraerts WP: Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network. Biochemistry 1998, 37:2070-2076. 22. Moroz LL, Gillette R, Sweedler JV: Single-cell analyses of nitrergic neurons in simple nervous systems. J Exp Biol 1999, 202:333-341. 23. Li LJ, Garden RW, Sweedler JV: Single-cell MALDI: a new tool for direct peptide profiling. Trends Biotechnol 2000, 18:151-160. 24. Rubakhin SS, Garden RW, Fuller RR, Sweedler JV: Measuring the peptides in individual organelles with mass spectrometry. Nat Biotechnol 2000, 18:172-175. 25. Amiri-Eliasi BJ, Fenselau C: Characterization of protein biomarkers desorbed by MALDI from whole fungal cells. Anal Chem 2001, 73:5228-5231.
681
26. Ryzhov V, Fenselau C: Characterization of the protein subset desorbed by MALDI from whole bacterial cells. Anal Chem 2001, 73:746-750. 27. ••
Fenselau C, Demirev PA: Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 2001, 20:157-171. Excellent review article on the different methodologies recently developed for the rapid protein profiling of intact microorganisms by mass spectrometry. 28. Caprioli RM, Farmer TB, Gile J: Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 1997, 69:4751-4760. 29. Chaurand P, Stoeckli M, Caprioli RM: Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry. Anal Chem 1999, 71:5263-5270. 30. Chaurand P, DaGue BB, Ma SG, Kasper S, Caprioli RM: Strainbased sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry. Biochemistry 2001, 40:9725-9733. 31. Chaurand P, DaGue BB, Pearsall RS, Threadgill DW, Caprioli RM: Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry. Proteomics 2001, 1:1320-1326. 32. Masumori N, Thomas TZ, Chaurand P, Case T, Paul M, Kasper S, Caprioli RM, Tsukamoto T, Shappell SB, Matusik RJ: A probasinlarge T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential. Cancer Res 2001, 61:2239-2249. 33. Garden RW, Sweedler JV: Heterogeneity within MALDI samples as revealed by mass spectrometric imaging. Anal Chem 2000, 72:30-36. 34. Stoeckli M, Farmer TB, Caprioli RM: Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 1999, 10:67-71.
Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections Pierre Chaurand, Sarah A Schwartz and Richard M Caprioli* MALDI MS imaging mass spectrometry can be used to map the distribution of targeted compounds in tissue sections with a spatial resolution currently of about 50 µm, providing important molecular information in many areas of biological research. After matrix application, a raster of a section by the laser beam yields ions from compounds in a tissue mass-tocharge range from 1000 to over 100 000. Two-dimensional intensity maps can then be reconstructed to provide specific molecular images of a tissue. Addresses Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-6400, USA *e-mail: [email protected] Current Opinion in Chemical Biology 2002, 6:676–681 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 August 2002 Abbreviations MALDI matrix-assisted laser desorption ionization TOF time-of-flight
Introduction Imaging mass spectrometry can be used to measure the spatial arrangement and relative concentration of compounds in biological samples, from elements to large proteins [1•]. One recently developed imaging technology utilizes matrix-assisted laser desorption ionization (MALDI) and time-of-flight mass spectrometry (TOF MS) to generate profiles and two-dimensional ion density maps of peptide and protein signals directly from the surface of thin tissue sections [1•,2••]. This allows specific information to be obtained on the local proteomic composition, relative abundance and spatial distribution of these components. Results from such imaging experiments yield a wealth of information, allowing investigators to measure and compare many of the major molecular components of the section to gain a better understanding of biological processes involved. MALDI mass spectrometry was first described in the late 1980s [3,4]. The sample preparation procedure involves mixing of a solution of a low molecular weight matrix molecule with analyte molecules (i.e. peptides and proteins) on the target plate. Evaporation of the solvent results in the formation of matrix-analyte co-crystals. Irradiation of these crystals by a short duration laser pulse initiates the desorption and ionization events, leading to the formation of singly charged protonated analyte molecular ions ([M+H]+). Recent improvements in time-of-flight technology [5,6] have allowed MALDI MS to become an extremely
accurate and sensitive tool for the detection, identification and characterization of peptides and proteins [7–9,10•,11,12,13•]. Proteins having molecular weights above 100 kDa can be detected for sample amounts in the femtomole range. Protein identification is accomplished from peptide maps following trypsin digestion and matching the results with theoretical digests of proteins in a database. For still higher confidence identification, the sequence analysis of one or more peptide fragments is obtained by tandem mass spectrometry, followed by sequence matching in the protein database [14–16].
Profiling proteins from blots of fresh tissue sections Over the past few years, several investigators have optimized protocols to generate protein profiles by MALDI MS from cells, groups of cells and small tissue sections immobilized on a target plate [17–26,27••]. Using MALDI MS, we have measured proteins in fresh tissues after transfer by contact blotting of the tissue on an active surface such as C18 [28] or a carbon-filled polyethylene membrane [29]. The latter is especially useful because it has both electrical conductivity and protein adhesion properties. Matrix was deposited on the blotted areas and, upon analysis, reproducible tissue-specific protein profiles were generated for a molecular range exceeding 100 kDa. Figure 1 presents the protein profiles obtained by contact blotting the different lobes of a mouse prostate on a polyethylene membrane. Contact blotting was performed for 5 min, covering the tissue samples with a glass slide to avoid dehydration. The blotted areas were rinsed with water to remove cell debris and tissue fragments as well as excess physiological salts and blood before matrix deposition. Data acquisition was performed in the linear mode under optimized delayed extraction conditions [5,6] on an Applied Biosystems DE-STR MALDI TOF mass spectrometer. After analysis of the different lobes of the prostate gland, very different and specific profiles containing over 300 individual signals, some at low intensities, were recorded in a m/z range up to 100 000. For example, the group of signals between m/z 21 800 and m/z 24 800, identified as different glycoforms of spermine-binding protein, have been found expressed at a much higher level in the ventral prostate lobe [30]. Similarly, the signal detected at m/z 75 094 has been detected at much higher intensity in the anterior prostate lobe. The profiles obtained after tissue blotting are extremely reproducible from animal to animal in the same strain line [29]. Using this new protein profiling approach, we have studied proteins present in sections of colon and prostate cancers, identifying several tumor-specific proteins [31,32].
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
677
Figure 1 Protein profiles obtained by MALDI MS after fresh tissue blotting on a polyethylene membrane for the mouse anterior (AP), dorsal (DP), lateral (LP) and ventral prostate (VP) lobes. α+ and β+ are α and β hemoglobin, respectively.
α+ β+ AP
DP
– –
LP
VP
6800
11 600
16 400
24 800
21 800
21 200
AP
75 094
Relative intensities
– –
DP
LP
VP 26 000
36 800
47 600
58 400
69 200
80 000
Mass (m/z) Current Opinion in Chemical Biology
Profiling and imaging of proteins directly from thin tissue sections Direct analysis of a thin tissue section by MALDI imaging mass spectrometry produces an array of signals in a mass range from 2 kDa to above 100 kDa, corresponding to hundreds of peptides and proteins present in the tissue. Figure 2 presents the protein profile obtained from a 12 µm human brain tissue section after application of sinapinic acid as matrix (saturated solution in a solution of 50/50/0.1 H2O/acetonitrile/trifluoroacetic acid, in volume). Thin sections, typically 12 µm, were cut at –15°C from snap frozen tissue samples using a cryostat. The sections were thaw-mounted on a target plate, allowed to dry in a dessicator for one hour and spotted with 200 nl of matrix before mass spectrometric analysis. In Figure 2, in the insert region alone in the m/z range from 5400 to 8400, over 60 individual mass signals were detected.
Principle of imaging mass spectrometry MALDI imaging mass spectrometry can be used to map the distribution of peptides and proteins directly from thin tissue
sections. Matrix is deposited uniformly over the section, and a raster of the tissue is performed over a predetermined twodimensional array or grid, generating a full mass spectrum at each grid coordinate. With commercially available instruments, the laser spot size on target can be reduced to about 50 µm in diameter after some fine-tuning of the optics, defining the actual imaging resolution. Each spectrum is the result of the average of 15 to 50 or more consecutive laser shots at each coordinate. From the intensity of a given m/z value monitored in each spectrum, a density map or image can be constructed. Virtually all of the signals detected from the section can be used to generate specific molecular weight images from a single raster of the tissue. The concept of MALDI-MS-based imaging mass spectrometry was introduced in 1997 by Caprioli and co-workers [28] to image fresh tissue slices either after coating the sample with matrix or after blotting the tissue slice on a target coated with C18 beads. In the latter case, a thin coat of matrix was deposited on the blotted area after removal of the tissue. Molecular ion
678
Analytical techniques
Figure 2
6086
7866
6971 5767 6664 6705
0 2000
7600
18 521
22 772
18 800
24 909
17 255
13 200
7800
7200
20 926
20
7383
6600
12 347
x3
6000
13 786 15 124 15 868
5358
5172
5400
40
7662
9956 10 625
8566
60
4039
% Intensity
80
Protein profile obtained by MALDI MS after matrix deposition (spotted with 200 nl of matrix) on a 12 µm human brain tissue section. Over 300 individual mass signals, some with low intensities, were detected.
7754
24 400
27 795
5651
4965
100
30 000
Mass (m/z) Current Opinion in Chemical Biology
images were successfully generated for rat splenic pancreas and for an area of the rat pituitary where over 50 different peptides were observed as well as some of their precursors, isoforms and metabolic fragments. Similarly, Garden and Sweedler [33] have mapped the intensity of standard peptides in MALDI samples prepared in a single drop of sample. Stoeckli et al. [34] developed a new imaging computer algorithm allowing both instrument control and data acquisition and processing for MALDI MS imaging of thin tissue sections. Using this approach, an image of a thin section of a human glioblastoma xenograph was obtained [2••].
Sample preparation and handling Careful sample preparation and handling is important to obtain high-quality images. For best results for high-resolution imaging, matrix must be deposited on the surface of the tissue in a homogeneous coat. Three conditions should be maintained to the greatest extent possible. First, the deposition process must not disperse or translocate proteins within the section. Second, the matrix solution must wet the tissue surface to form crystals, which contain co-crystallized proteins. Third, the crystal dimensions must be smaller than the image resolution. The following procedure is typical of sample preparation and can be used with most tissues. Immediately following
dissection, the tissue sections are loosely wrapped in aluminum foil and plunged into liquid nitrogen. Frozen tissue should be stored at –80°C until analysed. Twelvemicron sections are cut with a cryostat. This thickness is not critical and was chosen because of easy handling. The sample coating method that has proven to be the most successful thus far consists of air spraying matrix on the tissue. Matrix (saturated solution in a solution of 50/50/0.1 H2O/acetonitrile/ trifluoroacetic acid, in volume) is sprayed over the section using a commercially available glass spray nebulizer (similar to those used to coat thin-layer chromatography slides) connected to a nitrogen bottle (nebulizing gas) to minimize contamination. The coating procedure is as follows. About 500 µl of matrix are sprayed on the section at a distance of about 20 to 30 cm and the sample allowed to dry at room temperature (only a small fraction of the total volume is actually deposited on the section). It is important that the combination of the spray deposit and spray distance be optimized to avoid excessive wetting of the section, inevitably leading to protein delocalization. This spray cycle is repeated up to 10 times, allowing some air drying between cycles (30 s to 1 min) to generate a homogeneous crystal field. Sinapinic acid as matrix in the indicated solvent system typically yields crystals ranging in size from 5 µm to 25 µm in length. The quality of the mass
Figure 3 legend MALDI Imaging of a transversal rat brain section. (a) Survey profile with data acquisition taken randomly across the section. (b) Optical image of the section before matrix application. The general outlines of
the section as well as several features visible in the section have been delineated. (c)–(g) Ion density maps obtained at different m/z values. The section was imaged with a resolution of 180 µm.
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
679
Figure 3
(a) 100
7059
7532
6844, (d)
80 6714 6541
6909 7275
6062
5890 5632, (c)
% Intensity
60
6882
14117
40
7059 11 338, (e) 11 297
12 122 15 184
20
9969 15 807
3767 4960
18 393, (f) 21 882, (g)
0 2000
6600
11 200
15 800
20 400
25 000
Mass (m/z)
4 mm
(b) Optical image
(c) m/z 5632
optical
image
0
100%
a)
(d) m/z 6844
(e) m/z 11 338
(f) m/z 18 393
(g) m/z 22 920 Current Opinion in Chemical Biology
680
Analytical techniques
spectrometric data generated from this spray procedure is essentially the same as that generated using the drop deposition method.
spectrometry may also one day permit assessment of surgical margins at the cellular and molecular level.
Acknowledgements Application: imaging the rat brain The unique symmetry and well-characterized anatomy of brain allow it to be an excellent tissue model. Transversal rat brain sections were cut, thaw-mounted on the target plate (Figure 3b) and coated with matrix. First, a survey scan was performed with data acquisition taken randomly across the section to generate an average protein profile from this section (Figure 3a). Over 200 individual mass peaks were detected in a m/z range up to 40 000. Figure 3b presents an optical image of the brain section before matrix deposition. The general outlines of the section as well as several features visible in the section have been delineated. The section was scanned by acquiring 74 × 75 points with a resolution of 180 µm by averaging spectra produced by 15 laser shots using the automated imaging computer algorithm [34]. In this scan, the intensity of 45 different mass signals was monitored. Figure 3c–g presents five ion density maps obtained for different protein signals; some of these, in particular m/z 6844, have low intensities (see signals c–g in Figure 3b). As expected, some proteins were found to be very specific for a given brain region. This is particularly striking for the density maps of the proteins detected at m/z 5632 and m/z 18 393, which are almost ‘negatives’ of each other.
Conclusions Imaging mass spectrometry is still in an early stage and many developments and improvements in both sample preparation and handling as well as instrumentation are to be expected within the coming years. Items such as imaging acquisition speed, image resolution and data mining tools need to be further developed. Nonetheless, the potential of such a molecular imaging tool is enormous. The fundamental contributions of the technology in rapidly providing molecularweight-specific maps or images, at relatively high resolution and sensitivity, will provide a powerful tool for the investigation of cellular processes in both health and disease. Imaging mass spectrometry is of extraordinary benefit as a discovery tool where a tissue section can be investigated without necessarily knowing in advance what specific proteins have changed in a comparative study. Profile and image comparison between tissues allows researchers to easily identify differences in protein expression. Such experiments have already been performed comparing the profiles obtained for healthy and cancerous mouse colon tissue samples and, from these data, several cancer biomarkers were identified [31]. This study also provided valuable information on the relative concentration of proteins within the section. For clinicians, this will permit molecular assessment of tumor biopsies with the potential to identify sub-populations that are not evident based on the cellular phenotype determined microscopically. Further, it is not inconceivable that imaging mass
The authors thank Robert Weil (Department of Neurosurgery, Vanderbilt University Medical Center) for providing the human brain tissue section and Ariel Deutch (Departments of Psychiatry and Pharmacology, Vanderbilt University Medical Center) for providing the rat brain tissue sample. The authors also acknowledge funding by the National Institutes of Health (grant GM 58008).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1. •
Todd PJ, Schhaaff TG, Chaurand P, Caprioli RM: Organic ion imaging of biological tissue with SIMS and MALDI. J Mass Spectrom 2001, 36:355-369. Reference article describing the principle of imaging mass spectrometry and introducing its potential in cancer research. 2. ••
Stoeckli M, Chaurand P, Hallahan DE, Caprioli RM: Imaging mass spectrometry: a new technology for the analysis of protein expression in mammalian tissues. Nat Med 2001, 7:493-496. This tutorial article reviews the different techniques and methodologies employed for imaging organic compounds from thin tissue sections by mass spectrometry. 3.
Karas MH, Hillenkamp F: Laser desorption ionization of proteins with molecular masses exceeding 10000 Daltons. Anal Chem 1988, 60:2299-2301.
4.
Hillenkamp F, Karas M, Beavis RC, Chait BT: Matrix-assisted laser desorption ionization mass-spectrometry of biopolymers. Anal Chem 1991, 63:1193A-1202A.
5.
Brown RS, Lennon JJ: Mass resolution improvement by incorporation of pulsed ion extraction in a matrix-assisted laserdesorption ionization linear time-of-flight mass-spectrometer. Anal Chem 1995, 67:1998-2003.
6.
Vestal ML, Juhasz P, Martin SA: Delayed extraction matrix-assisted laser-desorption time-of-flight mass-spectrometry. Rapid Commun Mass Spectrom 1995, 9:1044-1050.
7.
Roepstorff P: Mass spectrometry in protein studies from genome to function. Curr Opin Biotechnol 1997, 8:6-13.
8.
Russell DH, Edmondson RD: High-resolution mass spectrometry and accurate mass measurements with emphasis on the characterization of peptides and proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Mass Spectrom 1997, 32:263-276.
9.
Lahm HW, Langen H: Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis 2000, 21:2105-2114.
10. Pandey A, Mann M: Proteomics to study genes and genomes. • Nature 2000, 405:837-846. This review article presents a clear and comprehensive overview of the role of mass spectrometry in modern proteomics. 11. McDonald WH, Yates JR: Proteomic tools for cell biology. Traffic 2000, 1:747-754. 12. Aebersold R, Goodlett DR: Mass spectrometry in proteomics. Chem Rev 2001, 101:269-295. 13. Godovac-Zimmermann J, Brown LR: Perspectives for mass • spectrometry and functional proteomics. Mass Spectrom Rev 2001, 20:1-57. Very detailed review article on the potential of mass spectrometry for the detection, identification and further characterization of peptides and proteins. 14. Mann M, Wilm M: Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 1994, 66:4390-4399. 15. Yates JR: Database searching using mass spectrometry data. Electrophoresis 1998, 19:893-900.
Imaging mass spectrometry Chaurand, Schwartz and Caprioli
16. Borchers C, Peter JF, Hall MC, Kunkel TA, Tomer KB: Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. Anal Chem 2000, 72:1163-1168. 17.
Gusev AI, Vasseur OJ, Proctor A, Sharkey AG, Hercules MH: Imaging of thin-layer chromatograms using matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 1995, 67:4565-4570.
18. Dreisewerd K, Kingston R, Geraerts WPM, Li KW: Direct mass spectrometric peptide profiling and sequencing of nervous tissues to identify peptides involved in male copulatory behavior in Lymnaea stagnalis. Int J Mass Spectrom 1997, 169:291-299. 19. Redeker V, Toullec JY, Vinh J, Rossier J, Soyez D: Combination of peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunodetection on single glands or cells. Anal Chem 1998, 70:1805-1811. 20. Jimenez CR, Burlingame AL: Ultramicroanalysis of peptide profiles in biological samples using MALDI mass spectrometry. Exp Nephrol 1998, 6:421-428. 21. Jimenez CR, Li KW, Dreisewerd K, Spijker S, Kingston R, Bateman RH, Burlingame AL, Smit AB, van Minnen J, Geraerts WP: Direct mass spectrometric peptide profiling and sequencing of single neurons reveals differential peptide patterns in a small neuronal network. Biochemistry 1998, 37:2070-2076. 22. Moroz LL, Gillette R, Sweedler JV: Single-cell analyses of nitrergic neurons in simple nervous systems. J Exp Biol 1999, 202:333-341. 23. Li LJ, Garden RW, Sweedler JV: Single-cell MALDI: a new tool for direct peptide profiling. Trends Biotechnol 2000, 18:151-160. 24. Rubakhin SS, Garden RW, Fuller RR, Sweedler JV: Measuring the peptides in individual organelles with mass spectrometry. Nat Biotechnol 2000, 18:172-175. 25. Amiri-Eliasi BJ, Fenselau C: Characterization of protein biomarkers desorbed by MALDI from whole fungal cells. Anal Chem 2001, 73:5228-5231.
681
26. Ryzhov V, Fenselau C: Characterization of the protein subset desorbed by MALDI from whole bacterial cells. Anal Chem 2001, 73:746-750. 27. ••
Fenselau C, Demirev PA: Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev 2001, 20:157-171. Excellent review article on the different methodologies recently developed for the rapid protein profiling of intact microorganisms by mass spectrometry. 28. Caprioli RM, Farmer TB, Gile J: Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 1997, 69:4751-4760. 29. Chaurand P, Stoeckli M, Caprioli RM: Direct profiling of proteins in biological tissue sections by MALDI mass spectrometry. Anal Chem 1999, 71:5263-5270. 30. Chaurand P, DaGue BB, Ma SG, Kasper S, Caprioli RM: Strainbased sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry. Biochemistry 2001, 40:9725-9733. 31. Chaurand P, DaGue BB, Pearsall RS, Threadgill DW, Caprioli RM: Profiling proteins from azoxymethane-induced colon tumors at the molecular level by matrix-assisted laser desorption/ionization mass spectrometry. Proteomics 2001, 1:1320-1326. 32. Masumori N, Thomas TZ, Chaurand P, Case T, Paul M, Kasper S, Caprioli RM, Tsukamoto T, Shappell SB, Matusik RJ: A probasinlarge T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential. Cancer Res 2001, 61:2239-2249. 33. Garden RW, Sweedler JV: Heterogeneity within MALDI samples as revealed by mass spectrometric imaging. Anal Chem 2000, 72:30-36. 34. Stoeckli M, Farmer TB, Caprioli RM: Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 1999, 10:67-71.