Direct Profiling of Proteins in Biological Tissue Sections by MALDI

Mass Spectrometry Research Center, Vanderbilt University School of Medicine, 824A Medical ... After treatment of the tissue blot with protease and sub...
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Direct Profiling of Proteins in Biological Tissue Sections by MALDI Mass Spectrometry Pierre Chaurand, Markus Stoeckli, and Richard M. Caprioli*

Mass Spectrometry Research Center, Vanderbilt University School of Medicine, 824A Medical Research Building I, Nashville, Tennessee 37232-6400

The direct profiling of proteins present in tissue sections for several organs of the mouse has been accomplished using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Fresh tissue was sectioned and blotted on a conductive polyethylene membrane. The dried membrane blot was coated with matrix, typically sinapinic acid, and directly analyzed in the mass spectrometer. Generally, well over 100 peptide/protein signals in the 2000-30 000 Da range were observed, with 3050 having relatively high signal intensities. Analysis of different areas of the same tissue gave remarkably similar mass spectra with greater than 90% homology. However, different parts of a segmented tissue, such as the proximal, intermediate, and distal colon, gave some unique protein signals. After treatment of the tissue blot with protease and subsequent MALDI MS analysis using postsource decay methods for peptide sequencing, some of the proteins were identified. The unique protein profiles measured from these tissue blots also showed differences from strain to strain of the mouse, with genetically similar strains having very similar patterns. One of the unique features of matrix-assisted laser desorption ionization (MALDI1,2) coupled to time-of-flight mass spectrometry (MS) is its ability to analyze relatively complex mixtures from samples that are not of high purity. Extracts from a wide variety of biological systems have been successfully analyzed, and in particular, peptide and protein mixtures have been identified from their measured molecular weights and partial sequence analysis. Although MALDI is relatively tolerant toward common contami* Corresponding author: (tel) (615) 322-4336; (fax) (615) 343-8372; (e-mail) [email protected]. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Karas, M.; Hillenkamp, F.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. 10.1021/ac990781q CCC: $18.00 Published on Web 10/29/1999

© 1999 American Chemical Society

nants such as buffers and salts,3 high concentrations of these can alter or inhibit cocrystallization between the matrix and the sample, resulting in low signal intensities for the analytes of interest. However, it has been demonstrated that washing of the sample after application of matrix can significantly increase signal intensities, presumably by eliminating much of the salt or other water-soluble contaminants. Other important considerations for obtaining optimal performance with MALDI MS are the methods used for sample preparation and nature of the target itself. Several investigators have purified peptides and proteins prior to mass spectrometry by electroblotting the samples onto targets using membranes such as poly(vinylidene fluoride) (PVDF)4-8 after separation and purification of the proteins by gel electrophoresis (SDS-PAGE). Other membranes, such as Nylon, polyethylene, or Teflon, have been used in different studies involving proteins9-11 and DNA12 in an attempt to further purify biological samples. In this procedure, samples were first blotted on membranes by simple passive contact and the membranes washed. The membranes were then directly mounted on mass spectrometry sample probes, matrix was added, and the samples were mass analyzed. (3) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (4) Eckerskorn, C.; Strupat, K.; Karas, M.; Hillenkamp, F.; Lottspeich, F. Electrophoresis 1992, 13, 664-665. (5) Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C., Lottspeich, F. Anal. Chem. 1994, 66, 464-471. (6) Mortz, E.; Vorm, O.; Mann, M.; Roepstorff, P. Biol. Mass Spectrom. 1994, 23, 249-261. (7) Gharahdaghi, F.; Kirchner, M.; Fernandez, J.; Mische, S. M. Anal. Biochem. 1996, 233, 94-99. (8) Vestling, M. M.; Fenselau, C. Mass Spectrom. Rev. 1995, 14, 169-178. (9) Ogorzalek Loo, R. R.; Mitchell, C.; Stevenson, T. I.; Loo, J. A.; Andrews, P. C. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 273-290. (10) McComb, M. E.; Oleschuck, R. D.; Manley, D. M.; Donald, L.; Chow, A.; ONeil, J. D. J.; Ems, W.; Standing, K.; Perreault, H. Rapid Commun. Mass Spectrom. 1997, 11, 1716-1722. (11) Worrall, T. A.; Cotter, R. J.; Woods, A. S. Anal. Chem. 1998, 70, 750-756. (12) Hung, K. C.; Ding, H.; Guo, B. Anal. Chem. 1998, 71, 512-521.

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Analysis of compounds obtained from fresh tissue using MALDI MS has been reported. The first approach utilized crude extracts of tissues followed by direct analysis by MALDI. For example, extracts of rabbit spleen were analyzed by Huff and coworkers and β-thymosin peptides were identified.13 Porcine cortex extracts were analyzed by Nilsson and Brodin identifying substance P-related peptides.14 Luo et al. investigated cardiac tissue extracts and have characterized di-adenosine polyphosphates.15 More recently, Lubman and colleagues analyzed malignant MCF10 Cala cells from culture and identified carcinogenesis-related proteins.16 Plant extracts have also been studied. Fructans have been characterized from onion bulb extracts by Stahl and co-workers17 while Sugui et al. studied anthocyanidins from sorghum plant tissue.18 In a second experimental approach, tissues were dissected and deposited in a small vial containing matrix, and the supernatant was analyzed.19 Jime´nez et al.20 analyzed neuropeptides secreted by neurons from freshwater snail. Van Strien et al.21 studied toad POMC gene-related neuropeptides secreted by the pituitary, and Jespersen et al.22 studied intrinsic disulfide bonds contained in the same neuropeptides. Other studies were performed by depositing matrix over freshly dissected tissues immobilized on metal surfaces. Dreisewerd and coworkers investigated peptides present in the single penis nerve of the pond snail.23 Redeker et al. studied the isoforms of procHH by direct peptide profiling of a single cas-producing cell.24 Jimenez and co-workers analyzed neuroendocrine cells in both invertebrate (pond snail) and vertebrates (xenopus and rat) as well as the giant neurons VD1 and RPD2 isolated from the brain of the mollusc Lymnaea stagnalis.25 Recently, Caprioli and co-workers used MALDI MS 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.26 In this second case, a thin coat of matrix was deposited on the blotted area after removal of the tissue. Molecular ion 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 their precursors, isoforms, and metabolic fragments. (13) Huff, T.; Muller, C. S. G.; Hannappel, E. FEBS Lett. 1997, 414, 39-44. (14) Nilsson, C. L.; Brodin, E. J. Chromatogr., A 1998, 800, 21-27. (15) Luo, J.; Jankowski, J.; van der Giet, M.; Gardanis, K.; Russ, T.; Vahlensieck, U.; Neumann, J.; Schmitz, W.; Tepel, M.; Deng, M. C.; Zidek, W.; Schluter, H. FASEB J. 1999, 13, 695-705. (16) Chong, B. E.; Lubman, D. M.; Rosenspire, A.; Miller, F. Rapid Commun. Mass Spectrom. 1998, 12, 1986-1993. (17) Stahl, B.; Linos, A.; Karas, M.; Hillenkamp, F.; Steup, M. Anal. Biochem. 1997, 246, 195-204. (18) Sugui, J. A.; Bonham, C.; Lo, S. C.; Wood, K. V.; Nicholson, R. L. Phytochemistry 1998, 48, 1063-1066. (19) Van Veelen, P. A.; Jimenez, C. R.; Li, K. W.; Wildering, W. C.; Geraerts, P. M.; Tjaden, U. R.; Greef, J. v. d. Org. Mass Spectrom. 1993, 28, 15421546. (20) Jimenez, C. R.; van Veelen, P. A.; Li, K. W.; Wildering, W. C.; Geraerts, P. M.; Tjaden, U. R.; van der Greef, J. J. Neurochem. 1994, 62, 404-407. (21) van Strien, F. J. C.; Jespersen, S.; van der Greef, J.; Jenks, B. G.; Roubos, E. W. FEBS Lett. 1996, 379, 165-170. (22) Jespersen, S.; Chaurand, P.; van Strien, F. J. C.; Spengler, B.; van der Greef, J. Anal. Chem. 1935, 7, 660-666. (23) Dreisewerd, S.; Kingston, R.; Geraerts, W. P. M.; Li, K. W. Int. J. Mass Spectrom. Ion Processes 1997, 169, 291-299. (24) Redeker, V.; Toullec, J. Y.; Vinh, J.; Rossier, J.; Soyez, D. Anal. Chem. 1998, 70, 1805-1811. (25) Jimenez, C. R.; Li, K. W.; Dreisewerd, S.; Spijker, R.; Kingston, R.; Bateman, R. H.; Burlingame, A. L.; Smit, A. B.; Minnen, J. v.; Geraerts, W. P. M. Biochemistry 1998, 37, 2070-2076. (26) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-60.

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More recently, Stoeckli et al. developed a new imaging computer algorithm allowing both instrument control and data imaging acquisition and processing for MALDI MS of thin tissue sections.27 Using the sample transfer to membrane approach, we have undertaken the study of fresh animal tissues without further sample purification. We have utilized the transfer capabilities of peptides and proteins from the surface of tissue samples to target membranes to generate a unique protein signal profile. The blotting of proteomic material to conductive polyethylene is demonstrated. Unique protein profiles have been generated as a function of both type of tissue used and location of the sample within a given tissue in the animal. Differences in protein profiles could also be observed between animals having different genetic backgrounds. Although our experiments were mainly focused on sample preparation and analytical methodologies, we also present approaches for protein identification in the complex mixtures that are found in tissues. We have identified some of the observed proteins using database searches and, in some cases, have confirmed this identification by on-target proteolytic digestion and subsequent sequence analysis by MALDI postsource decay (PSD).28-30 INSTRUMENTS AND METHODS Carbon-imbedded conductive polyethylene was used as a transfer membrane because of its protein blotting ability and its conductive properties. Conductive polyethylene was purchased in thin sheet format (80 µm) from Goodfellow Cambridge Ltd., Cambridge, England. The membrane was carefully fixed to the mass spectrometry sample target using double-sided conductive tape, (3M Co.). Special care was taken to mount the membrane in a perfectly flat manner to avoid trapping air bubbles. The conductive nature of both the membrane and the tape resulting in a low resistance (less than 50 kΩ) between the sample target plate (metallic) and the upper surface of the polyethylene film, thus greatly reducing charging of the sample surface.9 Typically, mass spectra obtained from samples desorbed from carbonimbedded polyethylene show a slight decrease in resolution with respect to metallic targets. This varies with particular samples and the resolution loss can be up to about 30%. Mouse colon tissue samples from six different animal strains were studied. Mice were dissected after being anesthetized using CO2 in a closed environment. The abdominal cavity was opened, and the large intestine was carefully removed and flushed with pure water to remove all the excrement contained inside. The large intestine was then opened along its longitudinal axis and spread out onto blotting paper. Sections from proximal, intermediate, and distal colon were carefully cut into 5-mm bands using scissors, and the inside face (large intestine epithelium) was applied on the transfer membrane for protein blotting. A microscope slide placed on top of the sections ensured proper contact with the membrane and minimized dehydration of the tissue. After 5 min, the pieces of tissue were removed and the polyethylene (27) Stoeckli, M.; Farmer, T. B.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1999, 10, 67-71. (28) Spengler, B.; Kirsh, D.; Kaufmann, R.; Jaeger, E. Rapid Commun. Mass Spectrom. 1992, 6, 105-108. (29) Kaufmann, R.; Spengler, B.; Luetzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910. (30) Chaurand, P.; Luetzenkirchen, F.; Spengler, B. J. Am. Soc. Mass Spectrom. 1999, 10, 91-103.

membrane was allowed to completely dry. Each blotted area was then rinsed with 20 µL of pure water for 30 s using a 20 µL automatic pipet to remove salts and any residual tissue fragments. When dried, the membrane was spotted with 1 µL of matrix (sinapinic acid from Sigma, at 10 mg/mL in acetonitrile/H2O, 1/1, v/v) on the areas blotted with tissues and allowed to dry in ambient atmosphere. Fresh tissue sampling and further analyses were always performed on the same day. Animals were used and handled according to University and NIH guidelines. On-target protein digestions were performed with trypsin (Sigma) at a concentration of 2 pmol/µL in 50 mM ammonium bicarbonate. The sample plate was heated to 37 °C and 1 µL of the trypsin solution was deposited onto the blotted areas prior to matrix deposition. The digestions were allowed to run for 30 min, regularly adding pure water to keep the sample wet. After 30 min, the samples were allowed to dry, and 1 µL of matrix was then deposited on the blotted areas without further washing. Samples were analyzed using a Perseptive DE-STR MALDI mass spectrometer (Perkin-Elmer Biosystems, Foster City, CA). The instrument was operated in the linear mode under optimized delayed extraction conditions.31 Mass spectra were randomly acquired over the entire sample surface using an average of 256 laser shots. Mass calibration was accomplished using the [M + H]+ ions of melittin (MW 2847.75, Sigma), bovine insulin (MW 5733.6, Sigma), and cytochrome c (horse heart, MW 12 360.1, Sigma). The calibration molecules were mixed with matrix (sinapinic acid) and deposited on the membranes either on or near the blotted areas. MALDI PSD analyses were performed on the same instrument after specific on-target proteolytic digestions. R-Cyano-4-hydroxycinnamic acid (10 mg/mL in acetonitrile/H2O, 1/1, v/v) was preferred as matrix for PSD analyses. Carbon-imbedded polyethylene is black, and MALDI sample visualization was a problem when illumination was performed at small angles with respect to the axis of the time-of-flight analyzer. To visualize the samples, light was introduced in the instrument using the window mounted above the ion source, illuminating the target plate at grazing angles. RESULTS Tissue Blotting Using Polyethylene Targets. To assess the effectiveness of blotting peptides and proteins to polyethylene, a proximal colon tissue sample from the Sweet Water River (SWR) mouse strain (Mus musculus) was applied to the membrane as described above. After rinsing, the blotted area was spotted with matrix and analyzed by MALDI MS. The rinsing water was centrifuged to precipitate tissue fragments. One microliter of the supernatant was deposited on the polyethylene membrane and was analyzed by MALDI MS after the addition of 1 µL of sinapinic acid. Simultaneously, an adjacent proximal colon sample was blotted for 5 min but was not washed. Matrix was deposited on blotted areas presenting no leftover tissue fragments due to the blotting process. This second blotted area was also analyzed by MALDI MS. Figure 1 presents three linear MALDI mass spectra obtained from the unwashed tissue sample (Figure 1a), the washed tissue sample (Figure 1b), and the rinsing water (Figure 1c). The three spectra are displayed with the same intensity scale. Ion signals in the mass-to-charge (m/z) range from 3000 to 30 000 (31) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003.

Figure 1. Positive linear MALDI mass spectra of proximal colon tissue samples blotted on polyethylene (a) not washed, (b) washed, and (c) of the rinsing water.

have been detected from both the tissue samples and the rinsing water. When comparing all three mass spectra of Figure 1, one can observe a relatively high degree of similarity although differences in relative intensity can be seen. Comparing Figure 1a and b shows that, after rinsing, the overall ion intensity has been decreased by a factor of about 1.5 across the displayed m/z range. From Figure 1c, it is clear that the rinsing procedure removes a significant amount of material from the surface of the membrane. Most of the molecules blotted on the membrane and expressed before and after washing are observed in the solution after rinsing. Comparing the profile of Figure 1c to the profile of Figure 1a and b, as a general trend, it appears that molecules of higher mass have a tendency to wash off the membrane with respect to molecules of lighter mass; i.e., the molecular weights measured in Figure 1c are skewed to the higher mass relative to Figure 1a. Although rinsing of the sample blot gives some loss of yield, the sample-to-sample reproducibility was found to be better after rinsing the membrane. Matrix deposited on remaining tissue fragments failed to generate adequate spectra. One of the critical parameters when blotting a fresh tissue sample is transfer time. Care must be taken to apply the tissue long enough to allow transfer but not so long that tissue dehydration occurs, leading to the attachment to the membrane of small tissue fragments when the tissue is removed. For example, for colon tissue, this can occur when blotting time exceeds 5 min. To estimate the time necessary for protein transfer, a blotting kinetic study was performed. Transfer times ranging from 10 to 300 s were successively measured, and it was found that it is necessary to apply the tissue sample for a minimum time period of at least 30 s to obtain a maximum transfer from the surface of colon tissue samples to the polyethylene membrane. The mass spectra obtained for blotting periods longer than 30 s were found to be virtually identical (more then 95% homology). Nature of Blotted Compounds. To verify that peptides and proteins preferentially bind to polyethylene membranes, an ontarget trypsin digestion of a blotted area was performed prior to matrix deposition. A distal colon tissue sample from the Black 6 strain (M. musculus) was blotted onto the membrane and trypsin Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

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Figure 2. Positive linear MALDI mass spectra of distal colon tissue samples blotted on polyethylene (b) before and (a) after proteolytic degradation by trypsin.

was applied to half of its surface. Two MALDI mass spectra, one from the digested area and one from the nondigested area, were recorded and compared. Results shown in Figure 2 indicate that tryptic digestion leads to the generation of lower m/z signals and the loss of higher m/z signals. This confirmed that most of the transferred (blotted) material on the polyethylene membrane was proteinaceous. Tissue Selection and Handling. To distinguish signals due to the presence of blood in our colon samples, a drop of blood was sampled by direct puncture of the heart or the vena cava. A drop of blood was directly deposited on the polyethylene membrane, allowed to dry, washed with 20 µL of water, spotted with matrix, and mass analyzed.32 Figure 3 presents the two mass spectra obtained (a) from blood sampled on the AKR mouse strain (M. musculus) and (b) from distal colon tissue sampled on the same ARK mouse strain. The hemoglobin R and β chains were clearly observed in both the blood (Figure 3a) and the distal colon tissue sample (Figure 3b) as singly and doubly charged molecular ions. In most of the different tissue samples that we have investigated, mass signals attributed to residual blood were detected. We observed that differences appeared in the m/z values of the R and β chains among the several mouse strains. For example, in the Black 6 strain the R and β chains appear at 14 981 and 15 618 Da, respectively, whereas in the AKR strain the R and β chains appear at 14 996 and 15 712 Da, respectively. These are genetic variants and their presence in tissue acts as internal standards. However, this must be done with caution since the exact mass may vary from strain to strain. Blood contamination could be minimized either by rinsing the blotted areas or by cutting larger area tissue samples (up to 5 mm in diameter in the case of colon samples), therefore limiting eventual bleeding on the blotted areas. In this case, sample washing was only performed in the center of the blotted surfaces avoiding resolubilization of any blood components derived from the cut edges of the tissue sample. (32) McComb, M. E.; Oleschuk, R. D.; Chow, A.; Ens, W.; Standing, K. G.; Perreault, H. Anal. Chem. 1998, 70, 5142-5149.

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Figure 3. Positive linear MALDI mass spectra of (a) blood and (b) distal colon tissue sampled on the same mouse and blotted on polyethylene. The singly and doubly charged ions of the R and β chains of hemoglobin are clearly detected in both samples.

Sample-to-Sample Reproducibility. To validate the use of polyethylene for tissue blotting, the sample-to-sample reproducibility of the pattern of blotted proteins was determined. From the same animal (F1 generation from a DBA/AKR cross), four adjacent distal colon tissue samples were blotted onto a polyethylene membrane and processed and analyzed as described earlier. The mass spectra recorded in each of the four blotted areas are presented in Figure 4. Ions signals in the m/z range from 200020 000 u were observed from the four areas. Using external calibration as described above, the mass assignment on each of the observed ions was found to be within two mass units from spectrum to spectrum over the entire m/z range. A detailed comparison of the spectra presented in Figure 4 revealed that the 33 most abundant mass signals were detected in all four profiles (doubly charged ions and ion signals due to matrix adducts were disregarded). Only four mass signals were expressed (at very low levels) in only three of the four spectra while very few mass signals (six) were expressed only once in one of the four spectra. In Figure 4, the relative intensities, with respect to the strong signal detected at m/z 9963, of 14 mass signals present in all four mass spectra with significant intensities were compared (doubly charged ions, ion signals due to matrix adducts, and blood components were disregarded). For each of the 14 mass signals, the coefficient of variation of the intensities was calculated. The coefficient of variation ranged from 0.0024 to 0.0273 with an average of 0.0112. Considering these statistics, the ion pattern was found to be highly reproducible in terms of both the observed signals and their relative intensities. A second set of measurements was performed comparing three individuals from the same strain. Differences, if any, in the protein profiles are indications of the level of genetic differences one can expect between individuals. Figure 5 compares the MALDI MS spectra of distal colon tissues, sampled on three different mice from the SWR strain. Calibration was performed using internal standards. When all three spectra were carefully analyzed, a total of 47 different mass signals with significant intensities could be detected in a mass range between 3000 and 20 000 Da (blood components, calibration standards, doubly charged ions, and ion

Figure 6. Positive linear MALDI mass spectra of (a) proximal, (b) middle, and (c) distal colon tissue samples from the same mouse blotted on polyethylene. A specific peptide and protein pattern can be observed for each part of the colon.

Figure 4. Positive linear MALDI mass spectra of four adjacent distal colon tissue slices, sampled from the same animal and blotted on polyethylene. Good sample-to-sample reproducibility can be observed.

Figure 7. Positive linear MALDI mass spectra of three distal colon tissue slices, sampled on three different mice of three different strains and blotted on polyethylene. The protein profile obtained from the SPRET/Ei strain differs from the patterns obtained from the SWR or A/J strains, indicating major genetic backgrounds.

Figure 5. Positive linear MALDI mass spectra of three distal colon tissue slices, sampled on three different mice of the same strain and blotted on polyethylene. Good strain-to-strain reproducibility can be observed.

signals due to matrix adducts were disregarded). Of these signals, 35 were detected in all three spectra, 6 were detected twice, and 6 were detected only once. Although in some cases differences in intensities could be observed (this is, for example, the case of the ion detected at m/z 15 995), the measured m/z values of the blotted proteins in these spectra are remarkably similar. Profiles from Different Sections of Colon. In these experiments, we examined differences in the expressed proteins in three

distinct parts of the mouse colon for several strains of mice. Tissue samples were collected in the proximal, middle, and distal colon. Figure 6 presents the three MALDI mass spectra of the proximal (Figure 6a), middle (Figure 6b), and distal (Figure 6c) colon sampled in a mouse of the AKR strain (M. musculus). The relative signal intensities among the three spectra have been retained. As a general trend, it was observed that some signals were present throughout the colon but at different relative intensities. Protein diversity was much higher in the proximal colon than in the middle or distal colon especially in the m/z range from 3000 to 10 000 Da. Some signals were only observed within the proximal colon, while for others, a diminishing gradient was observed from proximal to distal colon with strong intensities in the proximal colon, still detected in the middle colon, and absent in the distal colon. This is especially true for the protein signal expressed at m/z 28 230, which was detected at high concentration in the Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

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Table 1. List of Observed Molecular Ions from Figure 6b (after Subtraction of Signals Due to Blood Contamination) and Closest Match Found in Protein Databases for Mus musculus (Mouse) observed ions [M + H]+ (Da)

closest match in protein database for M. musculus [M + 1]a (Da)

∆M (Da)

3 493 4 983 5 135 5 709 5 756 6 048 6 312

3 496 MSZF75 (fragment) Krueppel-like zinc finger protein 4 982 disintegration 1 (fragment) 5 134 SP100 (fragment) 5 708 TP synthase  chain 5 757 LTR-AKT (fragment) 6 050 ATP synthase (fragment) 6 312 HTF9-A/RANBP1 (fragment) 6 312 cytochrome P450 6 803 protein kinase (fragment) 7 319 guanine nucleotide-binding protein, γ 5 subunit 7 798 signal transducer CD24 precursor 8 566 ubiquitin 8 987 cytochrome c oxidase VIIA-heart precursor 9 387 clone NCD36 line-1 element ORF1 (fragment) 9 622 MCF envelope protein (fragment) 9 624 MCZF47 (fragment) zinc finger 9 980 CRS4C precursor 10 079 neuronal death protein DP5 10 145 60S ribosomal protein L37A 10 147 CRS4C precursor 10 203 cochlear mRNA (clone 170A9S) 10 494 NADH-ubiquinone oxidoreductase chain 4L 10 494 PHXR4 putative per-hexamer repeat protein 4 10 497 cryptdin-7 precursor 10 828 CC chemokine ST38 precursor 10 829 PHXR3 putative per-hexamer repeat protein 3 10 832 HSP10 10-kDa heat shock protein 10 879 CREB C-AMP-responsive element binding protein 10 952 R-tubulin (fragment) 10 955 myoglobin 10 955 APOC3 apolipoprotein C-III precursor 11 056 calpactin I light chain 11 084 calgizzarin (EMAP) 11 344 apolipoprotein A-II 11 545 thioredoxin (ATL) 11 684 AGP R-1-acid glycoprotein (fragment) 11 687 POL protein (fragment) 11 810 IG κ chain V-V region UPC 61 11 811 IG κ chain V-V region J606 12 374 MIF macrophage migration inhibitory factor 12 376 ZFP72P (fragment) negative transcription regulator 12 440 periplakin (fragment) 12 440 Kar-associated protein 13 471 galanin precursor 13 680 ZFP77P (fragment) negative transcription regulator 13 771 serum amyloid A-1 protein precursor 13 774 serum amyloid A-3 protein precursor 15 995 FABP2, fatty acid binding protein 16 795 lysozyme C-type P precursor 16 793 prolactin-inducible protein homogue precursor

+3 -1 -1 -1 +1 +2 0 0 +3 +2 +2 0 +2 +2 -1 +1 +2 +1 0 +2 -1 -1 -1 +2 -2 -1 +2 +3 0 +3 +3 0 +1 +4 +3 -2 +1 -2 -1 0 +2 +3 +3 0 +1 -1 +2 0 +2 0

6 800 7 317 7 796 8 566 8 985 9 685 9 623 9 978 10 078 10 145 10 204 10 495 10 830 10 876 10 952 11 056 11 083 11 340 11 542 11 686 11 812 12 374 12 437 13 471 13 679 13 772 15 995 16 793 a

One mass unit mass was added to the database hits allowing direct comparison with the m/z values detected in the mass spectrum.

proximal colon and in trace amounts in the middle colon, but not detectable in the distal colon. For this particular protein, this behavior was observed for all of the mice strains studied. Although generally the protein diversity was greater in the proximal colon, a protein detected at m/z 9962 u was highly expressed in the distal colon with respect to the proximal or middle colon. When the five mice strains from the M. musculus species were compared over the same area of colon, most of the observed mass signals (about 70%) for the strains A/J, AKR, DBA/2, SWR, and Black 6 were found to be identical. The best signal correlation for these five strains was found to be in the mass range from 3000 to about 14 000 u. Again, strong variations in mass could be observed for the mass of the R and β chains of hemoglobin. Figure 5268

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7 displays three MALDI mass spectra of protein signals recovered from distal colon blots from three different strains of mice. The two top spectra display the profiles obtained from the SWR and A/J strains (M. musculus) whereas the bottom spectrum displays the profile obtained from the SPRET/Ei strain (Mus spretus). After each spectrum was carefully analyzed, it was found that 41, 51, and 80 signals were detected in the displayed mass range for the SWR, A/J, and SPRET/Ei strains, respectively. Comparing the SWR and A/J strains, one can see the relative similarities in the expressed proteins when comparing both mass and signal intensities. These two strains share as much as 33 mass signals (80% of the signals considering the SWR strain and 65% considering the A/J strain). These observations tend to demonstrate a fairly similar

genetic background between these two M. musculus strains.33 The mass spectrum generated from the SPRET/Ei strain was found to be very rich by its protein content in the displayed mass range. The SPRET/Ei strain shared 37 mass signals with the A/J strain (46%) and 21 signals with the SWR strain (25%). Furthermore, 21 signals were common to all of the three strains. When the general profile of the spectrum generated from the SPRET/Ei strain (M. spretus) was compared to the profiles generated from the SWR and A/J strains (M. musculus), major intensity differences could be observed. Even if some proteins have been detected in all of the compared strains, most of the proteins expressed in the SPRET/Ei strain were unique and very different with respect to the other strains. This observation plus the strong intensity differences observed when the profiles of the mass spectra were compared clearly demonstrates the differences in genetic background between the M. spretus mouse strain and the M. musculus strains of mice studied. Variations between the species have been shown at the DNA level. The evolutionary split between the M. spretus and M. musculus species dates back to about 2.5 million years (for comparison, the split between mouse and rat dates back about 12 million years) and about 1% of the noncoding DNA differs between the two species.33 The variation in the coding DNA is about 10-fold less with respect to the noncoding DNA. Considering this fact, it is surprising to find significant differences in the observed protein profiles. Although the reason is yet unknown, a possible explanation could be a relatively higher activity of one or more proteases in the SPRET/Ei strain, giving rise to higher abundance of low-mass protein fragments. Specific and unique protein profiles were obtained from localized fresh tissue using MALDI MS after blotting on a polyethylene membrane. The expressed protein profiles from each of the colon tissue samples studied were found to be unique even if very strong similarities were observed among individuals from the same strain. In addition, mass spectra from tissue blots of various organs such as prostate, epididymis, lymph node, liver, kidney, lung, spleen, and brain, obtained from species such as mouse, rat, and man, have been successfully obtained in our laboratory using MALDI MS. Unique profiles could be generated for each specific tissue, and major differences were found when similar tissues sampled on different species were compared. Protein Identification. Several strategies were employed in an attempt to identify some of the proteins expressed and blotted from the different tissue samples. The first was to search protein databases after having precisely determined the molecular weights of the proteins. This process, of course, is not a rigorous identification but does provide possible matches within a given molecular weight window. A protein database search was performed after precisely measuring the molecular weights of the signals observed in the mass spectrum displayed in Figure 3b (distal colon tissue from the AKR strain). Signals derived from matrix adduct, multiply charged ions, and blood components were disregarded. The search was performed with a mass tolerance of (3 u around the observed mass values in the mass range below 10 000 Da and with a mass tolerance of (5 mass units above 10 000 Da. Although a mass accuracy in the range of 10-4 can be expected, we have chosen to search with this wider tolerance for two reasons. First, for complex mixture analysis, overlaps of two (33) Silver, L. M. Mouse Genetics; Oxford University Press: New York, 1995.

Figure 8. PSD MALDI spectrum of a tryptic digest peptide obtained after on-target proteolytic degradation of a distal colon blot on polyethylene. The peptide total sequence SG(I/L)KVR could be retrieved from the spectrum analysis.

or more mass signals can influence the peak shape and centroid, thus giving a poorer mass measurement. Second, some modifications to the protein can give rise to small shifts of molecular weight, e.g., hydrolysis of an amide group to form the acid or reduction/oxidation of cysteine residues. Ninety-two mass signals were detected and labeled and database entries for 35 signals were made. Moreover, for some of the observed signals, multiple entries could be retrieved from the databases all within 1 or 2 mass units of the entered mass value. Database entries for these 35 protein mass signals are listed in Table 1. It is noted that database entries could not be retrieved for most of the strong intensity signals characteristic of the distal colon. Protein database searches have, however, significant limitations since molecular weights are generally calculated from amino acid sequences and do not take into account posttranslational modifications such as glycosylation, phosphorylation, acetylation, disulfide bond formation, and proteolytic processing. Another difficulty in working with databases is directly related to the particular animal species studied. For example, the number of protein entries for M. musculus (mouse) is relatively low with respect to other species. Database searching can, however, in some cases give strong indications of the nature of some of the proteins expressed in colon tissue. Confirmation of the exact nature of targeted proteins will require their isolation and further characterization. A second approach for the identification of blotted proteins was to obtain PSD fragment ion spectra from some of the lower mass peptides generated by on-target proteolytic digestions. Ontarget tryptic digestions have been performed on distal colon blots from several strains of mice. As a result of the digestions, peptides in a mass range between 500 and about 6000 Da were generated. PSD fragment ion spectra from several low-mass peptides have been successfully obtained. An example of such a spectrum is presented in Figure 8. The parent ion peptide is at mass 658.4 Da and has been detected in all of the studied mice strains. From the PSD spectrum of Figure 8, a full amino acid sequence could be deduced: NH2-SG(I/L)KVR-COOH with a high level of confidence. When submitted to protein databases (full database search), the sequence SGLKVR targeted one protein specific to Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

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the mouse: AP150 clathrin coat assembly protein. Clathrin, presumably at mass 49 654 Da according to its amino acid sequence, was not detected after tissue blotting on PE. Confirmation of the correct amino acid sequence would require its synthesis and the subsequent comparison of the PSD spectral profiles.34 The major drawback of targeting proteins by PSD MALDI is the absence of direct correlation between high-intensity protein signals and the intensities of the observed peptides generated after proteolytic degradation. One can only select and (partially) sequence digestion products giving sufficient fragmentation information in order to identify one or several proteins expressed by colon tissue. Second, partial (or total) sequence information retrieved from some of the PSD spectra might target high-mass proteins not detected in the original protein mass spectrum and eventually only detected by their fragments or degradation products in the mass range compatible with the polyethylene membrane. In these cases, even if some of the proteins have been identified, it will be difficult to make the relationship between these proteins and their fragments. CONCLUSIONS We have demonstrated that fresh tissues first blotted on conductive polyethylene generate unique protein profiles when (34) Flat, T.; Spengler, B.; Kalbacher, H.; Brossard, P.; Baier, D.; Kaufmann, R.; Bolt, P.; Metzger, S.; Bluggel, M.; Meyer, H. E.; Kurz, B.; Muller, C. A. Cancer Res. 1998, 58, 5803-5811.

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analyzed by MALDI MS. Fresh mouse colon tissue samples were analyzed and ion signals in a mass range from 2000 to 30 000 Da were observed. Sample-to-sample reproducibility was found to be excellent for tissue samples coming either from the same animal or from animals of the same strain. The protein expression specificity was demonstrated when the mass spectrum obtained after blotting the proximal, middle, and distal colon of the same animal was compared. Specific differences were also observed when the same region of the colon from several different strains of mice was compared, thus providing a clear signature of their different genetic backgrounds. ACKNOWLEDGMENT We sincerely thank Robert J. Coffey, Jr., and David W. Threadgill (Department of Cell Biology, Vanderbilt University) for providing the mouse colon samples. We thank the National Institutes of Health (Grants 1RO1GM 58008-01 and 1RO1GM 50529-05) and the Cancer Research Center of Vanderbilt University for financial support. Received for review July 15, 1999. Accepted October 6, 1999. AC990781Q