High‐resolution MALDI‐FT‐ICR MS imaging for the analysis of metabolites from formalin‐fixed,paraffin‐embedded clinical tissue samples

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin‐fixed, paraffin‐embedded (FFPE) human tissue samples. Using high‐resolution matrix‐assisted laser desorption/ionization Fourier‐transform ion cyclotron resonance mass spectrometry imaging (MALDI‐FT‐ICR MSI), we conducted a proof‐of‐principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI‐FT‐ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Molecular Morphology of the Chick Heart Visualized by MALDI Imaging Mass Spectrometry

Utilization of MALDI‐MS (matrix‐assisted laser desorption/ionization mass spectrometry) for tissue imaging is a relatively new proteomic technique that simultaneously maps the spatial distribution of multiple proteins directly within a single frozen tissue section. Here, we report the development of a methodology to apply MALDI tissue imaging to chick heart tissue sections acquired from fixed and paraffin‐embedded samples. This protocol produces molecular images that can be related to the high‐quality histological tissue sections. Perfused term chick hearts were fixed in acidic ethanol and embedded in paraffin wax. Tissue sections (15 μm) were collected onto conductive slides, deparaffinized with xylene, and transitioned into water with graded ethanol washes and allowed to air dry. In separate experiments, three different MALDI matrices were applied to chick heart tissue sections through repeated cycles from a glass nebulizer. Tissue sections were then analyzed by MALDI mass spectrometry using a raster step‐size of 75–100 μm, and molecular images for specific m/z ratios reconstituted. MALDI tissue imaging revealed spatially resolved protein signals within single heart sections that are specific to structures or regions of the heart, for example, vessels, valves, endocardium, myocardium, or septa. Moreover, no prior knowledge of protein expression is required as is the case for immunohistochemistry and in situ hybridization methodologies. The ability to simultaneously localize a large number of unique protein signals within a single tissue section, with good preservation of histological features, provides cardiovascular researchers a new tool to give insight into the molecular mechanisms underlying normal and pathological conditions. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections

Direct tissue profiling and imaging mass spectrometry (MS) provide a molecular assessment of numerous expressed proteins within a tissue sample. MALDI MS (matrix-assisted laser desorption ionization) analysis of thin tissue sections results in the visualization of 500 to 1000 individual protein signals in the molecular weight range from 2000 to over 200,000. These signals directly correlate with protein distribution within a specific region of the tissue sample. The systematic investigation of the section allows the construction of ion density maps, or specific molecular images, for virtually every signal detected in the analysis. Ultimately, hundreds of images, each at a specific molecular weight, may be obtained. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human non-small cell lung tumors, gliomas, and breast tumors. Interrogation of the resulting complex MS data sets using modern biocomputational tools has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These studies suggest that such proteomic information will become more and more important in assessing disease progression, prognosis, and drug efficacy. Molecular histology has been known for some time and its value clear in the field of pathology. Imaging mass spectrometry brings a new dimension of molecular data, one focusing on the disease phenotype. The present article reviews the state of the art of the technology and its complementarity with traditional histopathological analyses.     

Clinical applications of MALDI imaging using sliced sections of formalin-fixed paraffin-embedded tissues and longitudinal sliced hairs

MALDI-imaging MS (IMS) with MSMS analysis is a new powerful tool for the identification of not only disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections but also protein/peptides/drugs/medicine in fresh-frozen tissues. IMS is used to reveal the mass profiles and spatial distribution of proteins in tissue sections and/or digested peptides derived from deposited protein in pathologic organs and then MSMS analysis identifies the amino acid sequence of the detected proteins in the tissue section. Moreover, on-tissue digestion combined with the MALDI-IM-TOF-IMS approach allows a proteomics "bottom-up" strategy with clinical samples, especially perioperative isolated tissues and FFPE tissues conserved for a long time in a clinical sample bank. The mass barcode-like image (MBI) on a longitudinal sliced hair by IMS is used in the selected reaction monitoring mode for serially chronological monitoring and traceability every few hours after drug and medicine intake. The advances of quantitative MBI for sliced sections of hair allow a new universal standardized assessment of drugs and medicines throughout the drug history.     

Modern laser scanning microscopy in biology, biotechnology and medicine.

Laser microscopic techniques currently used in morphology and cell biology represent highly sensitive tools for detecting biomolecules within their natural environment. Use of the fluorescence-, reflectance- and transmission modes of confocal laser scanning microscopes (CLSM) equipped with He-Ne- and Ar+-ion lasers for CeIV and DAB based detection of endogenous or immunobound enzymatic activities in tissue sections (vibratome, cryostat, paraffin and semithin plastic sections) opens a wide range of interesting new possibilities in cellular and molecular biology. Increased resolution power, blur-free confocal imaging, higher sensitivity, optical sectioning capability and 3D-image analysis provide a large quantity of valuable information about biological objects specimens. The new infrared multiphoton laser scanning microscopy (NIR-LSM) is increasingly becoming the optical tool of choice for (a) fluorescence imaging of cellular and subcellular components with high spatial and temporal resolution, (b) fluorescence resonance energy transfer between physiologically relevant molecular species involving protein-protein interactions, (c) nanoprocessing within living cells and tissues, with varied applications in (d) photochemistry and (e) medical diagnostics as well. Both, CLSM and NIR-LSM as modern microscopical strategies are indispensable in basic research and will prove to be invaluable for clinical diagnostic studies and therapy in the near future.     

MALDI质谱成像技术在狂犬病病毒小鼠组织内定位研究中的应用

目的:建立基于MALDI质谱的狂犬病病毒小鼠脑组织内质谱成像实验方法,寻找狂犬病病毒脑组织内定位标记物。方法:制备冰冻切片,进行组织上原位酶解及基质覆盖,利用MALDI质谱扫描成像,以及FlexImaging 2.1软件分析,对差异肽段进行二级质谱鉴定。结果:初步鉴定出了四段狂犬病病毒肽段,可作为组织内狂犬病病毒定位标志物。结论:为进一步提高狂犬病病毒组织内定位的精确度奠定了研究基础。     

Medical molecular morphology with imaging mass spectrometry

Imaging mass spectrometry (IMS) is a two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules that does not need either separation or purification of target molecules and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix-assisted laser desorption/ionization (MALDI) is one of those most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. At present, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields.     

Proteomic profiling of hepatitis B virus-related hepatocellular carcinoma in China: a SELDI-TOF-MS study

Hepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality, but its underlying molecular mechanisms remain not well understood. High-throughput, proteomic techniques targeting unique biological molecules may provide novel insights into HCC pathogenesis and prognosis. In this study, we systemically investigated tissue biomarkers of HCC by using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technique. Proteomic spectra were generated from fresh tissues (26 HCC and 18 control cirrhotic liver) and analyzed by using Biomarker Wizard System. A total of 16 differential proteomic peaks were detected between HCC and cirrhotic liver tissues, and 11 between moderately and highly differentiated HCCs. The expression pattern of one proteomic peak was validated by immunohistochemistry. These molecules are potential candidate biomarkers for early diagnosis of and targeted therapy for HCC.     

Matrix-assisted laser desorption ionization mass spectrometry detection of proteins adsorbed in vivo onto contact lenses

Identification of the biomolecules that form the first adsorbed monolayer, which thus effect "interface conversion", in competitive adsorption from multicomponent biological solutions can be challenging because of limitations in mass resolution and sensitivity of established techniques. In this study matrix-assisted laser desorption ionization (MALDI) time of flight mass spectrometry is developed and applied as a novel surface analytical method to enable analysis of adsorbed multicomponent biomolecule layers directly on the biomaterial surfaces. We show that proteins adsorbed in vivo (on human eyes) on contact lenses can be detected rapidly and conveniently by the diagnostic highly resolved mass signals recorded by MALDI mass spectrometry. This new approach allows detection of minor (and major) proteinaceous constituents of biofouled layers at levels substantially below monolayer coverage. Identification is done by comparison with molecular masses of known proteins. Specifically, it is shown that in addition to lysozyme, other low molecular weight proteins adsorb from human tear fluid onto contact lenses; these proteins had not been detected in earlier studies using other techniques.     

De novo discovery of phenotypic intratumour heterogeneity using imaging mass spectrometry

An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intratumour clonal and phenotypic heterogeneity. However, the de novo identification of tumour subpopulations is so far both a challenging and an unresolved task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumour subpopulations. In this proof‐of‐principle study, spatially resolved, tumour‐specific mass spectra were acquired, using matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry, from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumour areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumour subpopulations. This was confirmed by linking the presence of these tumour subpopulations to the patients' clinical data. This revealed several of the detected tumour subpopulations to be associated with a different overall survival of the gastric cancer patients (p = 0.025) and the presence of locoregional metastases in patients with breast cancer (p = 0.036). The procedure presented is generic and opens novel options in cancer research, as it reveals microscopically indistinct tumour subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer, which may finally lead to new targeted therapies. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Rapid simultaneous detection of two orchid viruses using LC- and/or MALDI-mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption-ionization (MALDI) mass spectrometry are capable of providing molecular mass information on biological samples with high speed, accuracy and sensitivity. With mass spectrometry, identifying a virus based on the molecular weight of its coat protein is relatively simple and accurate. The technique can be applied to all viruses with known coat protein molecular weights. Using the LC/MS and/or MALDI, this paper describes rapid simultaneous detection of the two most prevalent orchid viruses, namely cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV). The coat protein molecular weights of CymMV and ORSV were detected accurately using an extract from 1 g of virus-infected Oncidium orchid flower. Because LC/MS and MALDI allow automated analyses of multiple samples with simple preparation steps, both techniques are ideal for rapid identification of viruses from a large number of samples. This is the first report on the application of LC/MS and/or MALDI for simultaneous detection of two plant viruses from an infected plant extract.     

MALDI imaging identifies prognostic seven-protein signature of novel tissue markers in intestinal-type gastric cancer

Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.     

Identifying prostate carcinoma by MALDI-Imaging

Prostate cancer has become one of the most common malignancies worldwide. Although lacking in specificity its diagnosis is still based partially on the serum-based test for prostate-specific antigen. As its pathogenesis has not yet been deciphered, the ongoing search for new and more reliable biomarkers remains a challenge to stratify disease onset and progression. Matrix-assisted laser desorption/ionization (MALDI)-Imaging is a promising technique to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles and correlation of the information with the histomorphological features of the same tissue section. This study describes the analysis of 22 prostate sections (11 with and 11 without prostate cancer) by MALDI-Imaging. Specific protein expression patterns were obtained for normal and cancerous regions within the tissue sections. Applying a 'support vector machine' algorithm to classify the cancerous from the non-cancerous regions, an overall cross-validation, a sensitivity and specificity of 88, 85.21 and 90.74%, respectively, was achieved. Additionally four distinctively overexpressed peaks were identified: 2,753 and 6,704 Da for non-cancerous glands, and 4,964 and 5,002 Da for cancerous glands. The results of this first clinical study utilizing the new technique of MALDI-Imaging underline its vast potential to identify candidates for more reliable prostate cancer tumor markers and to enlighten the pathogenesis of prostate cancer.     

Progression from cirrhosis to cancer is associated with early ubiquitin post‐translational modifications: identification of new biomarkers of cirrhosis at risk of malignancy

Cirrhosis is a lesion at risk of hepatocellular carcinoma (HCC). Identifying mechanisms associated with the transition from cirrhosis to HCC and characterizing biomarkers of cirrhosis at high risk of developing into cancer are crucial for improving early diagnosis and prognosis of HCC. We used MALDI imaging to compare mass spectra obtained from tissue sections of cirrhosis without HCC, cirrhosis with HCC, and HCC, and a top‐down proteomics approach to characterize differential biomarkers. We identified a truncated form of monomeric ubiquitin lacking the two C‐terminal glycine residues, Ubi(1‐74), the level of which increased progressively, from cirrhosis without HCC to cirrhosis with HCC to HCC. We showed that kallikrein‐related peptidase 6 (KLK6) catalysed the production of Ubi(1‐74) from monomeric ubiquitin. Furthermore, we demonstrated that KLK6 was induced de novo in cirrhosis and increased in HCC in parallel with accumulation of Ubi(1‐74). We investigated in vitro the possible consequences of Ubi(1‐74) accumulation and demonstrated that Ubi(1‐74) interferes with the normal ubiquitination machinery in what is likely to be a kinetic process. Our data suggest that de novo KLK6 expression during early liver carcinogenesis may induce production of Ubi(1‐74) by post‐translational modification of ubiquitin. Given the deleterious effect of Ubi(1‐74) on protein ubiquitination and the major role of ubiquitin machinery in maintenance of cell homeostasis, Ubi(1‐74) might severely impact a number of critical cellular functions during transition from cirrhosis to cancer. Ubi(1‐74) and KLK6 may serve as markers of cancer risk in patients with cirrhosis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Histology-directed matrix-assisted laser desorption/ionization analysis reveals tissue origin and p53 status of primary liver cancers

To date, protein profiles for hepatocellular carcinomas and cholangiocarcinomas have not been systematically evaluated and compared with each other in an unbiased way. Thirty-six hepatocellular carcinomas and adjacent normal tissue samples were analyzed using histology-directed, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Four cholangiocarcinomas and adjacent normal tissue samples were also evaluated. Tissue samples were sectioned at 10 μm, with 1-3 sections thaw-mounted on a conductive indium tin oxide-coated glass slide. Sinapinic acid was manually deposited on areas of each tissue section enriched by epithelial cells, either tumor or normal, and mass spectra were acquired using a MALDI-time of flight instrument. According to class prediction analysis, average prediction accuracy in test sets (composed of 18 hepatocellular carcinoma-normal pairs) ranged from 93.0 to 95.8%. Cholangiocarcinomas and hepatocellular carcinomas had different protein profiles, as evidenced by average prediction accuracy of >95% in the test set for all classifiers. Permutation P-values for 0.632 + bootstrap cross validated misclassification rates (at feature selection P < 0.001) were less than 0.05 for predicting p53 immunostaining status. We conclude that MALDI MS profiles may be useful in assisting with the diagnosis and the differential diagnosis of primary liver cancers.     

The demonstration of immunohistochemical biomarkers in methyl methacrylate-embedded plucked human hair follicles

Plucked human hair follicles have been proposed as a potential surrogate for tumour tissue for measuring the effect of drugs on pharmacodynamic biomarkers in drug intervention studies. We describe a new technique of embedding plucked hair follicles in the acrylic resin, methyl methacrylate, and the immunohistochemical demonstration of six potential biomarkers (Ki67, EGFR, phospho-p27, phospho-histone H3, phospho-MAPK and phospho-Rb) in de-plasticised sections. The advantages of this technique over those that have been used in support of clinical drug trials, such as skin and tumour biopsies, whole blood and whole hair samples is discussed.     

Pitfalls in diagnostic molecular pathology – significance of sampling error

Today, molecular diagnostic tests are widely used in clinical medicine with polymerase chain reaction (PCR)-based techniques being of particular interest. In tissue specimens, however, false-positive and false-negative results can be obtained if pathomorphological and processing aspects are not considered. We therefore studied the impact of tissue sampling in three widely used diagnostic tests: (1) assessment of clonality in B-cell non-Hodgkin's lymphoma, (2) analysis of microsatellite instability (MSI) in colorectal neoplasia, and (3) demonstration of mycobacterium tuberculosis. Tissue sections of routinely formalin-fixed and paraffin-embedded diagnostic specimens were taken, and the significance of sampling was systematically investigated using laser microdissection or processing of the entire section. PCR analyses were done according to standard protocols. False-positive pseudo-monoclonality was obtained in small gastrointestinal biopsies and in laser microdissected lymph follicles of non-neoplastic tonsils. False negativity (pseudo-stability) could be demonstrated in a case with hereditary rectal adenoma if whole tissue sections were taken without microdissection of the most dysplastic subareas. Demonstration of mycobacteria failed in tissue sections of a formalin-fixed lymph node that was positive after complete digestion or in fresh frozen material of the same patient. In diagnostic molecular pathology, sampling error is an important source of false-positive and false-negative results, particularly if disease- and tissue-specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity, are ignored.     

MALDI imaging as a specific diagnostic tool for routine cervical cytology specimens

Cervical squamous cell carcinoma (SCC) is among the most common malignancies in women worldwide. In developed countries routine cytology screening has dramatically reduced SCC mortality within the last three decades. High risk (HR) human papilloma virus (HPV) infection is the main causal factor in nearly 100% of invasive SCCs, in most cases of low grade squamous intraepithelial lesion (LSIL) and in nearly all cases of high grade squamous intraepithelial lesion (HSIL). Detection of HR-HPV DNA has been extensively evaluated for the triage of patients with low grade cytological abnormalities in order to identify those at greatest risk for underlying or developing HSIL or SCC. However, the vast majority of HR-HPV-positive precursor lesions will not progress to invasive cancer. A variety of other screening tools are available which aim to stratify clinically significant HPV infections and cytological alterations. Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a promising technology to assist in this endeavor. It delivers accurate mass spectrometric information of the sample's proteins and enables the visualization of the spatial distribution of protein expression profiles in correlation with histological features. In this study, 18 samples with Pap IIID or higher (>LSIL) and 14 samples with Pap I-II (WNL) were analyzed by MALDI imaging mass spectrometry (IMS). A genetic algorithm was applied to classify spectra resulting in an overall cross validation, sensitivity for Pap IIID and Pap?I-II of 83.7%, 88.9% and 78.6%, respectively. As this IMS based approach allows for unbiased and automated classifiction of cytological samples it appears to be a promising tool for stratification of cervical Pap smears.     

Development and use of biomarkers in oncology drug development

Successful development and use of biomarkers will improve the productivity of oncology drug development. Recognition of the importance of biomarkers for speeding drug development is reflected in the precise definitions and concepts proposed by an NIH Working Group to standardize terminology and promote a more coherent and systematic approach to the development and use of biomarkers. Potential clinical biomarkers of drug efficacy are often identified through pre-clinical studies or basic research. Identification of potential biomarkers for use in oncology is moving rapidly forward through continuing advances in clinical imaging technologies, especially molecular and functional imaging. Other rapid advances are a product of the growing availability of new scientific reagents for established technologies and of high-throughput genomic and proteomic technologies that can generate hundreds of potential biomarkers for further evaluation. In certain cases, conventional clinical diagnostic techniques or assays can be adapted for use in pre-clinical models to evaluate their ability to serve as biomarkers for predicting clinical responses to new drug candidates. Evaluation (pre-clinical and clinical) of a potential biomarker is often the longest stage of biomarker development, and standards for evaluation or validation depend on the intended use and stage of clinical development. Biomarkers verified for use in preclinical studies can be used to help select appropriate animal models and lead compounds. Biomarkers verified for use in clinical trials can confirm a drug's pharmacological or biological mechanism of action, guide protocol design, aid patient and dose selection, and help to minimize safety risks. Oncology drug development can be optimized by using a tiered set of clinical biomarkers that predict compound efficacy and safety with increasing confidence at each rise in tier thereby aiding corporate decision-making about advancing compounds. In oncology, a special class of extensively evaluated biomarkers of efficacy (surrogate endpoints) that generally correlate with desired clinical outcomes can be used as a basis for corporate decisions as well as for gaining accelerated provisional regulatory approval of a drug.     

Tissue‐based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer

Regional lymph node metastasis negatively affects prognosis in colon cancer patients. The molecular processes leading to regional lymph node metastasis are only partially understood and proteomic markers for metastasis are still scarce. Therefore, a tissue‐based proteomic approach was undertaken for identifying proteins associated with regional lymph node metastasis. Two complementary tissue‐based proteomic methods have been employed. MALDI imaging was used for identifying small proteins (≤25 kDa) in situ and label‐free quantitative proteomics was used for identifying larger proteins. A tissue cohort comprising primary colon tumours without metastasis (UICC II, pN0, n = 21) and with lymph node metastasis (UICC III, pN2, n = 33) was analysed. Subsequent validation of identified proteins was done by immunohistochemical staining on an independent tissue cohort consisting of primary colon tumour specimens (n = 168). MALDI imaging yielded ten discriminating m/z species, and label‐free quantitative proteomics 28 proteins. Two MALDI imaging‐derived candidate proteins (FXYD3 and S100A11) and one from the label‐free quantitative proteomics (GSTM3) were validated on the independent tissue cohort. All three markers correlated significantly with regional lymph node metastasis: FXYD3 (p = 0.0110), S100A11 (p = 0.0071), and GSTM3 (p = 0.0173). FXYD3 and S100A11 were more highly expressed in UICC II patient tumour tissues. GSTM3 was more highly expressed in UICC III patient tumour tissues. By our tissue‐based proteomic approach, we could identify a large panel of proteins which are associated with regional lymph node metastasis and which have not been described so far. Here we show that novel markers for regional lymph metastasis can be identified by MALDI imaging or label‐free quantitative proteomics and subsequently validated on an independent tissue cohort. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.