A streamlined mass spectrometry–based proteomics workflow for large-scale FFPE tissue analysis

Published on May 1, 2020in The Journal of Pathology7.996
· DOI :10.1002/PATH.5420
Fabian Coscia11
Estimated H-index: 11
(UCPH: University of Copenhagen),
Sophia Doll17
Estimated H-index: 17
(MPG: Max Planck Society)
+ 7 AuthorsMatthias Mann235
Estimated H-index: 235
(UCPH: University of Copenhagen)
Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Here, we describe an MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total work up took less than a day. We observed a moderate trend towards lower protein identifications in long-term stored samples (>15 years) but clustering into distinct proteomic subtypes was independent of archival time. Our results underline the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and prove the feasibility of analyzing large tissue cohorts in a robust, timely and streamlined manner. This article is protected by copyright. All rights reserved.
📖 Papers frequently viewed together
20133.52Amino Acids
7 Authors (Evelyne Maes, ..., Liliane Schoofs)
#1Stine Buch Thorsen (UCPH: University of Copenhagen)H-Index: 6
#2Irina GromovaH-Index: 40
Last. José M.A. Moreira (UCPH: University of Copenhagen)H-Index: 31
view all 8 authors...
The burden of colorectal cancer (CRC) is considerable—approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a ...
#1Mark A. Eckert (U of C: University of Chicago)H-Index: 60
#2Fabian Coscia (UCPH: University of Copenhagen)H-Index: 11
Last. Ernst Lengyel (U of C: University of Chicago)H-Index: 68
view all 20 authors...
High-grade serous carcinoma has a poor prognosis, owing primarily to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer1,2, but a systematic examination of both the tumour and stromal compartments is critical in understanding ovarian cancer metastasis. Here we develop a label-free proteomic workflow to analyse as few as 5,000 formalin-fixed, paraffin-embedded cells microdissected from each comp...
#1Tim StuartH-Index: 12
#2Rahul Satija (NYU: New York University)H-Index: 52
The recent maturation of single-cell RNA sequencing (scRNA-seq) technologies has coincided with transformative new methods to profile genetic, epigenetic, spatial, proteomic and lineage information in individual cells. This provides unique opportunities, alongside computational challenges, for integrative methods that can jointly learn across multiple types of data. Integrated analysis can discover relationships across cellular modalities, learn a holistic representation of the cell state, and e...
#1Christoph Wichmann (MPG: Max Planck Society)H-Index: 6
#2Florian Meier (MPG: Max Planck Society)H-Index: 20
Last. Matthias Mann (MPG: Max Planck Society)H-Index: 235
view all 6 authors...
: Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. Although achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run...
#1Sophia Doll (UCPH: University of Copenhagen)H-Index: 17
#2Florian Gnad (Cell Signaling Technology)H-Index: 46
Last. Matthias Mann (UCPH: University of Copenhagen)H-Index: 235
view all 3 authors...
: The concept of personalized medicine is predominantly been pursued through genomic and transcriptomic technologies, leading to the identification of multiple mutations in a large variety of cancers. However, it has proven challenging to distinguish driver and passenger mutations and to deal with tumor heterogeneity and resistant clonal populations. More generally, these heterogeneous mutation patterns do not in themselves predict the tumor phenotype. Analysis of the expressed proteins in a tum...
#1Simon J. Davis (University of Oxford)H-Index: 67
#2Connor Scott (John Radcliffe Hospital)H-Index: 6
Last. Roman Fischer (University of Oxford)H-Index: 50
view all 4 authors...
While nearly comprehensive proteome coverage can be achieved from bulk tissue or cultured cells, the data usually lacks spatial resolution. As a result, tissue based proteomics averages protein abundance across multiple cell types and/or localizations. With proteomics platforms lacking sensitivity and throughput to undertake deep single-cell proteome studies in order to resolve spatial or cell type dependent protein expression gradients within tissue, proteome analysis has been combined with sor...
: Compared to DNA, analysis of RNA is one step closer on the central dogma of biology to assessing cellular function. This makes it an extremely valuable target for research and clinical testing in nearly all areas of molecular biology. Most RNA molecules are ephemeral by nature. They exist as temporary intermediates, ostensibly enabling data transfer between the genome and the organism. Their ribose backbone renders them sensitive to simple degradation over time and they are the target molecule...
#1Hao Chi (CAS: Chinese Academy of Sciences)H-Index: 20
#2Chao Liu (CAS: Chinese Academy of Sciences)H-Index: 67
Last. Si-Min HeH-Index: 27
view all 19 authors...
Peptide identification in proteomics data is improved using an efficient open search engine.
#1Fabian Coscia (UCPH: University of Copenhagen)H-Index: 11
#2Ernst Lengyel (U of C: University of Chicago)H-Index: 68
Last. Marion Curtis (U of C: University of Chicago)H-Index: 7
view all 15 authors...
Summary Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free surviva...
#1Paul D. Piehowski (PNNL: Pacific Northwest National Laboratory)H-Index: 28
#2Vladislav A. Petyuk (PNNL: Pacific Northwest National Laboratory)H-Index: 38
Last. Karin D. Rodland (PNNL: Pacific Northwest National Laboratory)H-Index: 49
view all 16 authors...
Background Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed a...
Cited By29
#1Kevin A. KovalchikH-Index: 7
#2Qing Ma (U of O: University of Ottawa)H-Index: 1
Last. Isabelle SiroisH-Index: 11
view all 21 authors...
Mass spectrometry (MS)-based immunopeptidomics is maturing into an automatized, high-throughput technology, producing small- to large-scale datasets of clinically relevant MHC class I- and II-associated peptides. Consequently, the development of quality control (QC) and quality assurance (QA) systems capable of detecting sample and/or measurement issues is important for instrument operators and scientists in charge of downstream data interpretation. Here, we created MhcVizPipe (MVP), a semi-auto...
#1Qin Fu (Cedars-Sinai Medical Center)H-Index: 15
#2Christopher I. Murray (Cedars-Sinai Medical Center)H-Index: 17
Last. Jennifer E. Van Eyk (Cedars-Sinai Medical Center)H-Index: 86
view all 4 authors...
Sample preparation for mass spectrometry-based proteomics has many tedious and time-consuming steps that can introduce analytical errors. In particular, the steps around the proteolytic digestion of protein samples are prone to inconsistency. One route for reliable sample processing is the development and optimization of a workflow utilizing an automated liquid handling workstation. Diligent assessment of the sample type, protocol design, reagents, and incubation conditions can significantly imp...
#1Weiju Wu (Durham University)H-Index: 10
#2Noemi Lois ('QUB': Queen's University Belfast)H-Index: 47
Last. Roy A. Quinlan (Durham University)H-Index: 60
view all 10 authors...
Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-la...
#1Stephan Eckert (TUM: Technische Universität München)
#2Yun-Chien Chang (TUM: Technische Universität München)H-Index: 3
Last. Bernhard Kuster (DKFZ: German Cancer Research Center)H-Index: 84
view all 6 authors...
Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem m...
#1Xundan Zhou (Boston Children's Hospital)
#2Lei Qu (Boston Children's Hospital)
Last. Shaoli Wang (Boston Children's Hospital)
view all 6 authors...
Background. Traditional mass spectrometry detection methods have low detection efficiency for low-abundance proteins, thus limiting the application of proteomic analysis in the diagnosis of preeclampsia. Magnetic nanomaterials have good superparamagnetism and have obvious advantages in the field of biological separation and enrichment. Aim. The objective of this study is to explore the value of superparamagnetic iron oxide nanoparticles in the proteomic analysis of preeclampsia. Materials and Me...
#1Jorunn Stamnaes (University of Oslo)H-Index: 14
Celiac disease (CeD) is a prevalent intestinal disorder that only develops in genetically susceptible individuals when they mount a harmful CD4+ T-cell response towards gluten peptides. Intake of gluten leads to inflammation and remodeling of the small intestine with symptoms such as nausea and diarrhea. The only current treatment is a lifelong gluten free diet. The immunological basis for CeD is well characterized but the mechanisms that drive intestinal remodeling are still poorly understood. ...
#1Matthias Fahrner (University of Freiburg)H-Index: 4
#2Peter Bronsert (University of Freiburg)H-Index: 11
Last. Oliver Schilling (University of Freiburg)H-Index: 39
view all 5 authors...
Abstract null Colorectal adenocarcinomas (CRC) are one of the most commonly diagnosed tumors worldwide. Colorectal adenocarcinomas primarily metastasize into the liver and (less often) into the peritoneum. Patients suffering from CRC-liver metastasis (CRC-LM) typically present with a dismal overall survival compared to non-metastasized CRC patients. The metastasis process and metastasis-promoting factors in patients with CRC are under intensive debate. However, CRC studies investigating the prot...
#1Nina Ogrinc (university of lille)H-Index: 3
#2Pierre-Damien Caux (university of lille)H-Index: 1
Last. Zoltan Takats (university of lille)H-Index: 61
view all 12 authors...
Background null Formalin-fixed paraffin-embedded (FFPE) tissue has been the gold standard for routine pathology for general and cancer postoperative diagnostics. Despite robust histopathology, immunohistochemistry, and molecular methods, accurate diagnosis remains difficult for certain cases. Overall, the entire process can be time consuming, labor intensive, and does not reach over 90% diagnostic sensitivity and specificity. There is a growing need in onco-pathology for adjunct novel rapid, acc...
Many pathologies, including cancer, have been associated with aberrant phosphorylation-mediated signaling networks that drive altered cell proliferation, migration, metabolic regulation, and can le...
Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides ...
This website uses cookies.
We use cookies to improve your online experience. By continuing to use our website we assume you agree to the placement of these cookies.
To learn more, you can find in our Privacy Policy.