RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 2762–2766

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2119

Direct analysis of laser capture microdissected

endometrial carcinoma and epithelium by matrix-assisted

laser desorption/ionization mass spectrometry

Jingzhong Guo1, Terence J. Colgan2,3, Leroi V. DeSouza1, Mary Joe Rodrigues2,

Alexander D. Romaschin3,4 and K. W. Michael Siu1*1Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario,

Canada M3J 1P32Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X53Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College Street, Toronto, Ontario, Canada M5G 1L54Biochemistry, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4

Received 30 May 2005; Revised 26 July 2005; Accepted 26 July 2005

Direct analysis of laser capture microdissected malignant and normal endometrial epithelium

using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS)

was able to detect a number of proteins that are overexpressed in malignant epithelial cells. A total

of 16 physiologic and malignant endometrial samples were laser capture microdissected, including

four proliferative and four secretory endometria, and eight endometrioid adenocarcinomas. Two of

these proteins, at 10 834 and 10 843Da, likely correspond to calgranulin A and chaperonin 10, two

proteins that had previously been identified in endometrioid adenocarcinoma in whole tissue

homogenate by MS analysis. Direct analysis by MALDI-MS not only confirms that these proteins

are overexpressed in endometrial carcinoma, but also localizes them to the epithelial cells, the

expected cancer site. Copyright # 2005 John Wiley & Sons, Ltd.

Clinical proteomics aims to discover proteins of medical

importance, through an understanding of protein structure,

function, and expression.1 Tissue proteomics, one compo-

nent of clinical proteomics, seeks to identify differential pro-

tein expression in diseased versus normal tissue. Tissue

proteomics can be particularly challenging because samples

are highly heterogeneous with respect to cellular composi-

tion, secretions, and stage of disease.1–3 Nevertheless, tissue

proteomic analyses of human whole-tissue homogenates4–13

and microdissected cellular lysates following protein extrac-

tion in a buffer,2,14–28 using two-dimensional polyacrylamide

gel electrophoresis (2D-PAGE) and matrix-assisted laser des-

orption/ionization time-of-flight mass spectrometry (MAL-

DI-TOFMS), surface-enhanced laser desorption/ionization

mass spectrometry (SELDI-MS), or isotope-coded affinity

tags followed by multiple stages of chromatography and

online tandem mass spectrometry (MS/MS), have been suc-

cessful in identifying some proteins which appear to be asso-

ciated with particular diseases.

The potential of applying proteomic techniques for the

direct analysis of microdissected tissue and tissue sections is

attractive to analytical chemists as well as to surgical

pathologists. Direct tissue analysis is attractive to the former,

as little or no sample preparation and separation are required,

while, to the latter, it preserves protein location information, a

much desired piece of information for disease diagnosis.

MALDI-MS ‘imaging’ of intact biological tissues was reported

to detect a large number of peptides and signals.29,30 The first

application of MS directly to microdissected mouse and

human tissue samples, including normal and malignant

breast epithelium, reported over 40 peaks that differed

significantly in intensity between invasive mammary carci-

noma and normal breast epithelium.31 The status of tissue

profiling, including profiling of laser capture microdissection

(LCM)-procured cells, by MS, has recently been reviewed.30

Most studies reported advances in techniques or methodol-

ogies and the observations of MS peaks that permit differ-

entiation between disease and normal states, but no specific

protein identifications.29–38 Protein markers were identified

in a few studies after extensive purification via multi-step

separation, partial sequencing, and MS identification.39–41

Here we report findings of a feasibility study in which we

use direct MALDI analysis of LCM-procured endometrial

cells to localize protein markers previously identified in

whole-tissue homogenates of cancerous human endome-

trium.10,12 This is the first study in which direct MALDI

analysis is used to validate and localize cancer markers

discovered with whole tissue homogenates and in a rarely

examined but common women’s cancer, i.e., endometrial

cancer. The American Cancer Society estimates that in 2005

there will be 40 880 new cases of cancer of the uterine body,

of which the vast majority will be endometrial cancer.42

Copyright # 2005 John Wiley & Sons, Ltd.

*Correspondence to: K. W. M. Siu, Department of Chemistry,York University, 4700 Keele Street, Toronto, Ontario, CanadaM3J 1P3.E-mail: kwmsiu@yorku.caContract/grant sponsor: A Canadian Cancer Society NationalCancer Institute of Canada (NCIC) Research Grant.

EXPERIMENTAL

Tissue consent and samplesEndometrial tissue and tumor samples were retrieved from a

dedicated, research in-house endometrial tissue bank. The

consenting and tissue-banking procedures had been

approved by the Research Ethics Boards of York University,

Mount Sinai Hospital, University Health Network, and

North York General Hospital. All tissues were obtained

with patients’ consent, and snap-frozen in liquid nitrogen

within 15–20 min of devitalization at the time of hysterect-

omy. Samples were then stored in a freezer at �708C prior

to use. In each case, the endometrium was classified by one

of the authors (TJC) as carcinoma (including subtype) or phy-

siologic (including secretory and proliferative endometria).43

A total of 16 physiologic and malignant endometrial cases

were the subject of this study, including eight endometrioid

adenocarcinomas, and four proliferative and four secretory

endometria. Histopathologic classification was performed

using routine surgical pathology hematoxylin and eosin sec-

tions from paraffin blocks.

Direct analysis of microdissected samplesusing MALDI-MSFrozen samples were retrieved from the tissue bank and

placed into a standard cryostat (Leica, Chicago, USA) at

�208C. After embedding in optimal cutting temperature

(OCT) compound, the tissue was placed on the chuck and fro-

zen. The tissue block was sectioned at 7 mm and sections

mounted on pre-cleaned glass slides. The slides were then

transported on dry ice, returned to the �708C freezer, and

stored until LCM was performed. Upon removal from the

freezer, the slides were transported on dry ice, ensuring

that thawing longer than 30 s was avoided. The slides were

then fixed and dehydrated as follows: 75% ethanol (30 s), dis-

tilled and deionized water (30 s), 75% ethanol (30 s), 95% etha-

nol (30 s), 3� 100% ethanol (60 s each), and finally 2� xylene

(240 s each). The slides were then air-dried for 15 min in a

fumehood and then stored in a desiccator until LCM.

Microdissection was performed on unstained sections using

a PixCell II (Arcturus, Mountain View, CA, USA) at room

temperature by a dedicated research histotechnologist (MJR)

under the supervision of a board-certified anatomic patholo-

gist (TJC). Carcinoma or endometrial glandular epithelium

was dissected from surrounding stroma (see Fig. 1) using a 7.5

or 15mm infrared laser beam, until approximately 80% of the

area of the Capsure Macro LCM cap (Arcturus) was filled.

Caps were then placed into an Eppendorf tube, and stored at

�708C until direct MALDI-MS was performed.

For MS analysis, the thermoplastic film was freed from the

LCM cap using forceps, and placed onto the MALDI plate

using double-sided tape. A matrix solution of a 10-fold

dilution of saturated sinapinic acid in 60% acetonitrile and

0.3% trifluoroacetic acid was applied over the cells. The

matrix was placed on the microdissected cells using a capil-

lary connected to a pump-driven syringe under microscopic

visualization. The final volume of the matrix delivered varied

from 0.1 to 1mL.

The direct LCM samples were analyzed using a

hybrid QqTOF mass spectrometer (QSTAR XL, Applied

Figure 1. (a) A proliferative endometrial gland in an

unstained, fixed and dehydrated cryosection as visualized

under a PixCell II; (b) following LCM, the gland has been

removed leaving a defect in the tissue section; and (c) the

microdissected gland residing on the CapSure cap.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2762–2766

Direct MALDI-MS of endometrial carcinoma and epithelium 2763

Biosystems/MDS Sciex), fitted with a MALDI interface.

Internal mass calibration was performed using insulin

(5733 Da), cytochrome c (12 361 Da) and myoglobin

(16 951 Da) as calibrants.

RESULTS

Typical direct MALDI mass spectra of LCM-procured epithe-

lial cells from two carcinomas (top) and two physiologic

endometrial tissues (bottom) are shown in Fig. 2. The spectra

of the carcinoma cells (C1 and C6) differ from those of the

physiologic endometrial epithelial cells (N4 and N6) by the

consistent presence of a number of peaks: (1) a cluster of

three peaks centered at 3443 Th; (2) a peak at 10 091 Th; (3)

two partially resolved peaks labeled as 10 835 Th in the spec-

tra; and (4) a peak at 11 651 Th. These features are shown more

clearly in Fig. 3 in limited-range mass spectral windows

around these peaks.

Analyses of the eight carcinoma samples allow determina-

tion of the precision of the determination of the protein

masses, assuming singly protonated proteins: 3370.5�0.0 Da;

3441.5� 0.0 Da; 3485.5� 0.0 Da; 10 090.1� 0.5 Da; 10 833.8�0.7 Da; 10 842.2� 0.4 Da; and 11 650.4� 0.9 Da. In accordance

with the better resolution attainable for smaller protein ions,

the precision of the mass measurements, a standard deviation

of �0.0 Da, was much better for the cluster of three smallest

proteins/peptides than for the other larger proteins. Compar-

isons between carcinoma and physiologic endometrial sam-

ples revealed three abundant common proteins: 11 305.2�1.3 Da; 13 774.1� 0.4 Da; and 14 002.6� 0.7 Da. The standard

deviations were all smaller than the generic expected

accuracy of �2 Da for proteins around 10 kDa.

Table 1 shows a summary of the abundances of these

proteins in the eight carcinoma and the eight physiologic

endometrial LCM-procured epithelial cells analyzed. As

before,10,12 the abundances are expressed semi-quantita-

tively in a scale of 0 to 5þ based on the signal/noise (S/N)

ratio because of the inherently poor repeatability (precision)

of MALDI analyses. It is apparent that the abundances of all

marker proteins in the carcinoma samples are higher than

those in the physiologic endometrial samples, irrespective of

whether absolute or relative abundances are considered. The

relative abundances shown are those normalized against the

common protein at 11 305 Da; this choice of the protein is non-

critical as relative abundances calculated using the other two

common proteins are similar.

The discriminating proteins at 10 833.8� 0.7 Da and

10 842.2� 0.4 Da have masses that are comparable to the

molecular weights of two potential cancer markers identified

in homogenates of cancerous endometrial tissues,10,12

namely, calgranulin A (10 834 Da) and chaperonin 10

(10 843 Da). It is noteworthy that calgranulin A is one of two

proteins in the Swiss-Prot database that have a molecular

weight of 10 834� 2 Da; calgranulin A was identified as such

by the MS/MS spectra of three of its tryptic peptides.12

DISCUSSION

Direct MALDI analysis of microdissected cells has opened

up a new avenue for research. Whether this technique is

applicable to all tissues is currently uncertain. For example,

laser capture dissection for global protein expression

profiling may not be suitable to all tissue types, if the desired

cell type cannot be accurately identified and isolated, or if

LCM does not provide cellular enrichment.44 Thus, studies

of the technical feasibility of applying this novel technique

to a variety of human tumors and tissues seem appropriate.

This is the first study of normal and malignant endometrial

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Figure 2. Spectra from direct MALDI analysis of two microdissected carcinoma samples (C1 and

C6: top), and two microdissected physiologic endometrial samples (N4 and N6; bottom). Note the

cluster of peaks in the carcinoma samples around 3443, 10 091, 10 835 and 11 651Th.

2764 J. Guo et al.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2762–2766

epithelium using direct MALDI analysis of microdissected

cells. In addition, unlike previous studies on direct MALDI

analysis, our results on differential expression confirm pro-

tein identities made for endometrial tissue homogenates10,12

and localizes the proteins to pure epithelial cells, the expected

cancer site.

Our study indicates that direct MALDI-MS analyses of

LCM-procured endometrial epithelial cells, under the

conditions described herein, do provide reliable spectra.

Furthermore, a comparison of the MS protein expression

profiles of endometrial carcinoma with physiologic endo-

metrial epithelium reveals several additional proteins of

increased expression in the malignant tissues (Figs. 2 and 3,

Table 1). Identification of these proteins will require extensive

microdissection to scale up for extensive chromatographic

purification, trypsin digestion and MS/MS, steps that can

only be taken after our throughput in LCM is increased by

very many fold (previous protein identifications were

performed with 500 mg of proteins).10,12

To preserve sensitivity, this study avoided the use of any

histological staining of tissues submitted to direct MALDI;

mass spectra were obtained from unstained tissue sections.

The PixCell II provided sufficient visualization for accurate

dissection of endometrial tissue. Our initial attempts at

MALDI analysis of microdissected stained cells confirmed

the experience of others, that such staining does impair the

acquisition of consistent MS spectra.31 Although there is a

theoretical concern that some soluble proteins may be lost in

the dehydration and fixation steps, comparisons of spectra

from untreated tissue with those from tissue treated with

alcohol and water washes have shown no significant loss of

protein.31

One advantage of direct MALDI analysis of microdissected

cells is that few cells are required to obtain abundant protein

Table 1. Abundances of protein markers: absolute abundance (relative abundance normalized to 11 305Da)

Case 3371 Da 3442 Da 3486 Da 10 090 Da 10 842 Da 10 834 Da 11 650 DaC1 3þ (3þ) 3þ (4þ) 3þ (3þ) 3þ (2þ) 2þ (2þ) 3þ (3þ) 3þ (3þ)C2 0 (0) 0 (0) 0 (0) 5þ (5þ) 1þ (2þ) 1þ (2þ) 4þ (5þ)C3 1þ (1þ) 1þ (2þ) 1þ (1þ) 3þ (3þ) 3þ (4þ) 1þ (1þ) 2þ (3þ)C4 4þ (4þ) 4þ (5þ) 5þ (5þ) 5þ (4þ) 5þ (5þ) 4þ (4þ) 5þ (5þ)C5 1þ (1þ) 1þ (1þ) 1þ (1þ) 1þ (1þ) 2þ (2þ) 1þ (1þ) 1þ (2þ)C6 3þ (3þ) 3þ (3þ) 3þ (2þ) 2þ (1þ) 3þ (3þ) 3þ (3þ) 3þ (2þ)C7 1þ (1þ) 1þ (1þ) 1þ (1þ) 1þ (1þ) 2þ (2þ) 1þ (2þ) 1þ (1þ)C8 5þ (5þ) 5þ (5þ) 3þ (5þ) 2þ (2þ) 3þ (4þ) 5þ (5þ) 2þ (2þ)N1 0 (0) 0 (0) 0 (0) 0 (0) 1þ (1þ) 0 (0) 1þ (1þ)N2 0 (0) 0 (0) 0 (0) 0 (0) 1þ (2þ) 0 (0) 1þ (1þ)N3 0 (0) 0 (0) 0 (0) 0 (0) 2þ (2þ) 1þ (1þ) 1þ (1þ)N4 0 (0) 0 (0) 0 (0) 0 (0) 1þ (1þ) 0 (0) 1þ (1þ)N5 0 (0) 0 (0) 0 (0) 0 (0) 1þ (2þ) 0 (0) 1þ (1þ)N6 0 (0) 0 (0) 0 (0) 1þ (1þ) 1þ (1þ) 0 (0) 1þ (1þ)N7 0 (0) 0 (0) 0 (0) 1þ (1þ) 1þ (1þ) 1þ (1þ) 1þ (1þ)N8 0 (0) 0 (0) 0 (0) 1þ (1þ) 1þ (1þ) 1þ (1þ) 1þ (1þ)

C, carcinoma samples; N, physiologic endometrial samples.1þ, S/N¼ 2–5; 2þ, S/N¼ 5–10; 3þ, S/N¼ 10–15; 4þ, S/N¼ 15–20; 5þ, S/N¼ 20–100.

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Figure 3. Spectra from direct MALDI analysis of a microdissected carcinoma, zoom-in

spectra for each marker protein of one sample (C1). Three small marker proteins are

shown in the 20Th windows on the top, and other large marker proteins are shown in the

100Th windows at the bottom.

Direct MALDI-MS of endometrial carcinoma and epithelium 2765

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2762–2766

signals and the consequent spectrum.31 In comparison, one

study of esophageal carcinoma used 50 000 microdissected

cells for differential protein expression using 2D-PAGE.45 In

our experience, acquisition of spectra typically required an

accumulation of 100 scans in less than 2 min, and usually

consumed less than 20%, or approximately 2000, of the

available endometrial epithelial cells on the cap. A MALDI

spectrum with a S/N ratio� 5 can be obtained from an

accumulation of 100 shots from as few as approximately 600

cells. Firing of a second batch of 100 laser shots at these cells

produces a MALDI spectrum of lower S/N ratio, but

typically still retaining all the critical spectral features.

CONCLUSIONS

Direct MALDI analysis of microdissected endometrial phy-

siologic and malignant epithelium reveals differentially

expressed proteins, two of which (chaperonin 10 and calgra-

nulin A) have previously been identified in endometrial tis-

sue homogenates. In addition, the results in this study

localize these proteins to endometrial epithelial cells, the

expected cancer site.

AcknowledgmentsThe authors gratefully acknowledge the support and coop-

eration of their surgical and clinical laboratory collaborators,

including Drs. K. Joan Murphy (University Health Network),

Wusun Paek (Mount Sinai Hospital), and Titus Owalabi, Ali

Qizilbash and Dennis MacDonald (North York General Hos-

pital). This study was supported by the Canadian Cancer

Society in the form of a National Cancer Institute of Canada

(NCIC) Research Grant. Infrastructural support was pro-

vided by the Canadian Foundation for Innovation (CFI), the

Ontario Innovation Trust (OIT), and the Ontario Research

and Development Challenge Fund (ORDCF). Additional

support by Genome Canada is also acknowledged.

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