Technical Note pubs.acs.org/jpr
Tryptic Peptide Reference Data Sets for MALDI Imaging Mass Spectrometry on Formalin-fixed Ovarian Cancer Tissues Stephan Meding,† Karina Martin,† Ove J. R. Gustafsson,† James S. Eddes,† Sandra Hack,† Martin K. Oehler,‡ and Peter Hoffmann*,† †
Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, SA 5005, Adelaide, Australia Department of Gynaecological Oncology, Royal Adelaide Hospital, SA 5000, Adelaide, Australia
‡
S Supporting Information *
ABSTRACT: MALDI imaging mass spectrometry is a powerful tool for morphology-based proteomic tissue analysis. However, peptide identification is still a major challenge due to low S/N ratios, low mass accuracy and difficulties in correlating observed m/z species with peptide identities. To address this, we have analyzed tryptic digests of formalin-fixed paraffin-embedded tissue microarray cores, from 31 ovarian cancer patients, by LC−MS/MS. The sample preparation closely resembled the MALDI imaging workflow in order to create representative reference data sets containing peptides also observable in MALDI imaging experiments. This resulted in 3844 distinct peptide sequences, at a false discovery rate of 1%, for the entire cohort and an average of 982 distinct peptide sequences per sample. From this, a total of 840 proteins and, on average, 297 proteins per sample could be inferred. To support the efforts of the Chromosome-centric Human Proteome Project Consortium, we have annotated these proteins with their respective chromosome location. In the presented work, the benefit of using a large cohort of data sets was exemplified by correct identification of several m/z species observed in a MALDI imaging experiment. The tryptic peptide data sets generated will facilitate peptide identification in future MALDI imaging studies on ovarian cancer. KEYWORDS: ovarian cancer, tissue, FFPE, proteomics, liquid chromatography, MALDI imaging
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FFPE tissue proteomes.14 New preparative methods for bottom-up proteomics have enabled the identification of proteins previously inaccessible from FFPE cells and tissue.14−16 However, these approaches are lysate based and thus destroy the morphological integrity of the analyzed tissues. Hence, the histopathological analysis has to be performed on adjacent tissue sections, increasing the risk of overlooking key morphological tissue features. Tissue lysis can be circumvented using MALDI imaging mass spectrometry (MALDI imaging), which combines morphological tissue analysis with in situ mass spectrometry.17 At first, MALDI imaging was limited to native, cryo-preserved tissues. Recent developments in MALDI imaging now enable researchers to investigate molecular profiles directly from FFPE tissue sections.17−23 These new protocols render clinical tissue archives accessible to MALDI imaging. Analyzing large patient cohorts using whole tissue sections is time-consuming and susceptible to technical variation due to the preparatory effort of analyzing every section individually.
INTRODUCTION Ovarian cancer is the fifth most common cause of cancer death among women.1 A more fundamental understanding of the molecular events associated with ovarian cancer progression, invasion, metastasis, therapy response and relapse may aid in the anticipated development of early detection techniques, novel therapies or individualized treatment regimes.2−5 Formalin-fixed paraffin-embedded (FFPE) patient tissues are the gold standard for tissue-based clinical diagnostics and research.6−8 This is primarily due to easy storage, handling and processing of FFPE tissue as well as its compatibility with most in situ techniques, such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).9 The virtually universal utilization of FFPE tissues has led to collection of vast clinical archives, complete with clinical meta-data such as survival and treatment response. These archives are valuable sample sources for retrospective studies seeking to elucidate the mechanisms underlying ovarian cancer.10 Insightful studies rely on the application of robust protocols for analyzing these FFPE tissues.11,12 Accessing proteins directly from FFPE tissue for MS analysis has proven difficult due to the extensive inter- and intraprotein cross-linking induced by formalin treatment.13 These cross-links have, until recently, limited the exploration of © 2012 American Chemical Society
Special Issue: Chromosome-centric Human Proteome Project Received: October 23, 2012 Published: December 10, 2012 308
dx.doi.org/10.1021/pr300996x | J. Proteome Res. 2013, 12, 308−315
Journal of Proteome Research
Technical Note
of similar data sets will be more common in the future. The manuscript describes how these data sets can be tested for quality and annotated to contribute them to the C-HPP.
Tissue microarrays (TMA) were established in clinical research to address restrictions due to analysis time and technical reproducibility issues.24 A TMA comprises multiple cores (typically
