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Top-down capability is a major advantage of MALD imaging, allowing the analysis of intact endogenous peptides and proteins. However, performing a tryptic digest directly on the tissue can be beneficial for several reasons:

Proteins too large for the mass range of a typical MALDI imaging experiment can be digested with trypsin and the resulting peptides analyzed. Fragmentation and identification of tryptic peptides is

easier than for endogenous peptides. Tryptic digestion is the method of choice for the analysis

of formalin-fixed paraffin embedded (FFPE) tissue.To be compatible with MALDI imaging, the localization of peptides generated by a tryptic digest procedure must match with the original localization of the undigested and diffusion across the tissue must be prevented. In addition, compatibility with subsequent histological staining is important, especially for the analysis of tumor sections.The ImagePrep, with its ability to deposit minute droplets onto tissue in a controlled environment is well suited to perform enzymatic digests on tissue. Here we describe the use of the ImagePrep for this purpose.

Introduction

In recent years, MALDI imaging has been established as a straightforward way to analyze tissue sections, allowing

Application Note # MT-98

Spatially Resolved Tryptic Digest on Tissue using the ImagePrep

untargeted molecular analysis in combination with histology. Some of the greatest challenges in MALDI imaging can be resolved by on-tissue digestion, these are: The standard MALDI imaging approach is limited to the

analysis of fresh-frozen tissue. Especially in pathology, large collections of formalin fixed paraffin embedded (FFPE) tissue are available, and usually come with a complete patient history, which makes the interpretation of results in a clinical context much easier. Access to and handling of FFPE is also much easier, but cross-linking of proteins in the fixation process renders FFPE tissue essentially useless for direct MALDI imaging. The mass range observed in direct MALDI imaging

experiments usually does not extend beyond 30kDa. MALDI is generally biased towards lower molecular weight, and biological tissues contain a complex mixture of proteins and other analytes. Although there are many reports of identified proteins in

MALDI imaging experiments, identification even of small endogenous peptides by MS/MS analysis directly off the tissue can be challenging. Interference from a highly complex background, unexpected modifications, unspecific cleavage sites complicate database searches. Often, identification requires purification of the target protein, and is not possible directly off the tissue.

Enzymatic digests provide a possible solution to all of these challenges: Tryptic digests have been reported to allow the analysis

of FFPE tissues by MALDI imaging [1-4]. Tryptic peptides of proteins are within the range

accessible by MALDI imaging, and may serve as an indicator of the original protein’s presence in the tissue Fragmentation and identification of tryptic peptides is

usually more straightforwardA drawback of tryptic digestion on tissue is that the initial comlexity of analytes is tremendously increased, since each digested protein usually produces numerous tryptic peptides, all of which fall in to a similar mass range. In addition, information usually accessible by top-down approaches, such as ongoing proteolytic processes in the tissue, can be obscured by the tryptic digestion.The ImagePrep, with its ability to deliver small droplets onto tissue sections in a controlled manner, is well suited for on-tissue digests as shown in the application note.

Experimental

A specific protocol and ImagePrep methods have been developed for on-tissue digeste. A detailed, illustrated step-by-step description is available in the Bruker Daltonics download area for registered users.In brief, rat brain sections were washed twice for two minutes each in ethanol (70%) and once for two minutes in ethanol (100%). 200μl trypsin solution (100 ng/μl, in 50mM NH4HCO3, pH 7.5-8.5) was loaded into the spray head as described in the protocol and the tryptic digestion ImagePrep method was executed. This method repeatedly sprays small amounts of trypsin solution onto the tissue and incubates the tissue for two hours in a humid environment. During the procedure, part of the section was covered with a coverslip to prevent digestion and act as negative control.

After digestion, the entire tissue section was coated with a-Cyano-4-hydroxycinnamic acid using the standard ImagePrep method. After matrix coating, slides were dip-washed in cold ammonium phosphate buffer (10mM (NH4)H2PO4, monobasic in 0.1% TFA).The MALDI analysis was performed using an ultraflex III mass spectrometer operating in reflectron mode. Acquisition and visualization was done with the flexImaging 2.1 software package. Selected peptides were analyzed in MS/MS mode and identified by a Mascot database search.After the MALDI imaging experiment, matrix was removed by washing in 70% Ethanol and an H&E staining of the tissue was performed according to standard protocols.

Results

In figure 1 average spectra of the digested and the undigested brain areas are shown. The effect of the tryptic digestion is immediately apparent. Most prominent signals visible in the undigested control have disappeared as a result of trypsination, and numerous signals have appeared in the mass range typical for tryptic peptides. This demonstrates a clear effect of the enzymatic treatment.Figures 2 a-f show the localization of different peptides in the section and the respective annotated MS/MS spectra allowing their identification. The distributions of the identified peptides are confined to anatomically defined structures in the brain, e.g. Cam-kinase II alpha in the hippocampus and cortex, and hemoglobin near major blood vessels. This proves that the spatial distribution of the tryptic peptides remains intact during the digestion procedure.In many cases, evaluation of MALDI imaging results is only possible based on an H&E stained tissue section. For an unambiguous interpretation it is highly desirable to perform the H&E staining on the same tissue section as the MALDI

Validation of effective digestion

Fig. 1: Comparison of average spectra of undigested and digested area of rat brain sample

digested area

undigested control

digested area

undigested control

* undiges ted HC C A average\0_L 15\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000m/z

1585 1590 1595 1600 1605 1610m/z

* diges ted HC C A average\0_L 9\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000

digested area

undigested control

digested area

undigested control

* undiges ted HC C A average\0_L 15\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000m/z

1585 1590 1595 1600 1605 1610m/z

* diges ted HC C A average\0_L 9\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000

digested area

undigested control

digested area

undigested control

* undiges ted HC C A average\0_L 15\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000m/z

1585 1590 1595 1600 1605 1610m/z

* diges ted HC C A average\0_L 9\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000

digested area

undigested control

digested area

undigested control

* undiges ted HC C A average\0_L 15\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000m/z

1585 1590 1595 1600 1605 1610m/z

* diges ted HC C A average\0_L 9\1\1S R ef R aw

1000 1500 2000 2500 3000 3500 4000 4500 5000

Localization of identified peptides in the brain

Fig. 2: Spatial distribution and MS/MS spectra of selected peptides. Endogenous peptides are detected in the undigested control (upper half) and tryptic peptides in the digested part (lower half).

1497 Da1497 Da

m/z200 400 600 800 1000 1200 1400 1600

0

2

4

6

8

10

12

14

16

18

20

Abs. Int. * 1000a Q Gb Q G P P Qy R I P G P K P

100.993a 1

129.011b 1

175.035y 1

Q

158.009a 2

185.996b 2

288.036y 2

283.040b 3

385.104y 3

P

380.086b 4

442.114y 4

508.106b 5

Q

610.200y 6

K

705.214a 7

733.190b 7

764.210y 8

887.253b 9

861.295y 9

989.308y 10

1214.489y 12

1311.641y 13

I

1496.751y 15

R PQK y12b6

PPQK y13b6

KPPGP y10b10

PPGPAGPI y9b14

Synapsin-1

Tryptic fragment

1730 Da1730 Da

m /z250 500 750 1000 1250 1500 1750 0

1

2

3

4

5

6

7

8

9

Abs . Int. * 1000a I T A Ab A A E Ay R H S I H K L A

86.037a 1

175.030y 1

I

187.002a 2

215.039b 2

312.027y 2

T

257.985a 3

286.031b 3

399.040y 3

329.031a 4

357.072b 4

512.093y 4

486.066b 5

E

557.126b 6

795.277y 6

642.178a 7

932.345y 7

1060.351y 8

K

907.358a 9

1173.491y 9

H1244.475

y 10P

1218.371b 11

W1555.645

b 14

1683.653a 15

1729.954y 15

R

LK y9b8

CaM-kinase II alpha chain

Tryptic fragment

2141 Da2141 Da

m /z500 1000 1500 2000

0

10

20

30

40

50

Abs . Int. * 1000a T Q N P V V H F Fb D E N P V V H F F K N Ty R P T V I N K F F H V N E D Q T

73.957a 1

174.983y 1

T

201.970a 2

229.946b 2

271.964y 2

Q

344.907b 3

372.932y 3

445.881a 4

473.870b 4

471.978y 4

559.923a 5

587.903b 5

585.016y 5

N

656.944a 6

684.976b 6

699.017y 6

P

756.021a 7

784.026b 7

827.147y 7

V 855.146a 8

883.099b 8

974.181y 8

992.190a 9

1020.137b 9

1121.266y 9H

1139.244a 10

1167.184b 10

1258.358y 10

F

1286.387a 11

1314.368b 11

1357.383y 11

1442.407b 12 1556.514

b 13

1553.581y 13

1641.494a 14

1667.696y 14

I

1768.678b 15

Myelin basic protein (MBP)

Tryptic fragment

1573 Da1573 Da

m/z250 500 750 1000 1250 1500 1750

0

2

4

6

8

10

12

14

16

18

Abs. Int. * 1000a I G G G E Y G Eb G G E Y G E E A L Qy R Q L A E E G Y E G G H G G I

86.028a 1

175.026y 1

I

142.990a 2

171.033b 2

303.043y 2

416.122y 3

337.070a 4

365.076b 4

487.149y 4

H

394.068a 5

422.092b 5

616.164y 5

451.112a 6

479.107b 6

745.206y 6

580.149a 7

608.140b 7

802.240y 7

E

743.201a 8

771.168b 8

965.248y 8

Y

800.154a 9

828.188b 9

1094.122y 9

929.239a 10

957.232b 10

1151.274y 10

1086.218b 11

1208.325y 11

1157.311b 12

1345.474y 12

1270.438b 13

1402.491y 13

1398.513b 14

1459.612y 14

Q

1572.656y 15

R

GG y14b3

GGEYGEEA y11b12

Hemoglobin subunit alpha-1/2

Tryptic fragment

1755 Da1755 Da

m /z250 500 750 1000 1250 1500 1750

0

2

4

6

8

10

12

14

16

18

Abs . Int. * 1000a S Q F R Qb F R K F Q K K K Qy S Q S K K K Q F Q S

60.071a 1

106.038y 1

S

188.035a 2

216.021b 2

234.050y 2

Q

335.092a 3

363.086b 3

321.032y 3

F

491.182a 4

519.137b 4

R

647.257b 5

449.130y 5

K

766.308a 6

794.337b 6

577.192y 6

894.364a 7

922.367b 7

705.297y 7

1050.483b 8

833.383y 8

1178.491b 9

961.367y 9

1306.626b 10

1349.741a 11

1392.814y 12

1521.862b 13

1539.973y 13

1622.114a 14

1650.069b 14

1668.048y 14

1754.980y 15

QFRK y14b5

KKKAGS y8b13

KFQKKKA y11b11

Brain-specific polypeptide PEP-19

Endogeneous peptide

Hemoglobin subunit beta-2

1326 Da

m/z200 400 600 800 1000 1200 1400

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs. Int. * 1000a L Yb L V V Y Py Q T W P

85.972a 1

113.953b 1

L

213.086b 2

338.042y 2

V 284.049a 3

312.110b 3

466.108y 3

447.091a 4

475.108b 4

567.129y 4

Y

572.173b 5

753.272y 5

850.340y 6

W

R

1324.688y 10

YP y7b5

TQ y4b8

PWT y6b7

Endogeneous peptide

a)

b)

c)

d)

e)

f)

imaging experiment [5]. Figure 3 shows the result of an H&E stain following tryptic digestion and MALDI imaging analysis. Although the section is not preserved as well as in the control (which was subjected to MALDI analysis but not trypsin digestion), the tissue remains sufficiently intact to see the nuclei and histological features even after digestion.

Conclusion

We have presented an easy method for tryptic digestion on tissue sections. The method requires no modification of the

ImagePrep and can be executed in a straight-forward and automated fashion. The method is efficient and provides a good digestion yield, but preserves the tissue to allow H&E staining after digestion and MALDI imaging. The localization of peptides is preserved throughout the protocol and tryptic peptides can be identified directly from the tissue using LIFT MS/MS. This application widens the scope of MALDI imaging considerably, especially because it enables the analysis of FFPE tissue.

References

[1] Lemaire R, Desmons A, Tabet JC, Day R, Salzet M, Fournier I. Direct analysis and MALDI imaging of formalin-fixed, paraffin- embedded tissue sections. J. Proteome Res. 2007 ,6, 1295-1305[2] Stauber J, Lemaire R, Franck J, Bonnel D, Croix D, Day R, Wisztorski M, Fournier I, Salzet M. MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting J. Proteome Res 2008,7, 969-978[3] Ronci M, Bonanno E, Colantoni A, Pieroni L, Di Ilio C, Spagnoli LG, Federici G, Urbani A. Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations. Proteomics 2008, 8,3702-3714[4] Groseclose MR, Massion PP, Chaurand P, Caprioli RM. High-throughput proteomic analysis of formalin-fixed paraffin- embedded tissue microarrays using MALDI imaging mass spectrometry. Proteomics 2008 , 8,3715-3724[5] Walch A, Rauser S, Deininger SO, Höfler H. MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology. Histochem Cell Biol. 2008, 130,421-434.

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Authors

Martin Schürenberg, Sören Deininger, Michael Becker, Bruker Daltonics, Bremen, Germany.

Acknowledgements

We thank Eckhard Friauf and Bernd Kaltwaßer, University of Kaiserslautern, for providing the rat brain sample.

Keywords

MALDI Imaging

tryptic digest

on-tissue peptide ID

peptide identification

H&E staining

FFPE tissue

Tissue imaging

Instrumentation & Software

ultraflex III TOF/TOF

ImagePrep

For research use only. Not for use in diagnostic procedures.

H&E staining on digested tissue

digested area

undigested control

500 µm

200 µm

Fig. 3: H&E staining of rat brain after tryptic digestion and MALDI imaging