A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples.

Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.

A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue.

BACKGROUND: Pathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. RESULTS: We demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings. CONCLUSIONS: The findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

A High-Throughput PIXUL-Matrix-Based Toolbox to Profile Frozen and Formalin-Fixed Paraffin-Embedded Tissues Multiomes.

Large-scale high-dimensional multiomics studies are essential to unravel molecular complexity in health and disease. We developed an integrated system for tissue sampling (CryoGrid), analytes preparation (PIXUL), and downstream multiomic analysis in a 96-well plate format (Matrix), MultiomicsTracks96, which we used to interrogate matched frozen and formalin-fixed paraffin-embedded (FFPE) mouse organs. Using this system, we generated 8-dimensional omics data sets encompassing 4 molecular layers of intracellular organization: epigenome (H3K27Ac, H3K4m3, RNA polymerase II, and 5mC levels), transcriptome (messenger RNA levels), epitranscriptome (m6A levels), and proteome (protein levels) in brain, heart, kidney, and liver. There was a high correlation between data from matched frozen and FFPE organs. The Segway genome segmentation algorithm applied to epigenomic profiles confirmed known organ-specific superenhancers in both FFPE and frozen samples. Linear regression analysis showed that proteomic profiles, known to be poorly correlated with transcriptomic data, can be more accurately predicted by the full suite of multiomics data, compared with using epigenomic, transcriptomic, or epitranscriptomic measurements individually.

High-Resolution Genotyping of Formalin-Fixed Tissue Accurately Estimates Polygenic Risk Scores in Human Diseases.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in biobanks and pathology archives are a vast but underutilized source for molecular studies on different diseases. Beyond being the "gold standard" for preservation of diagnostic human tissues, FFPE samples retain similar genetic information as matching blood samples, which could make FFPE samples an ideal resource for genomic analysis. However, research on this resource has been hindered by the perception that DNA extracted from FFPE samples is of poor quality. Here, we show that germline disease-predisposing variants and polygenic risk scores (PRS) can be identified from FFPE normal tissue (FFPE-NT) DNA with high accuracy. We optimized the performance of FFPE-NT DNA on a genome-wide array containing 657,675 variants. Via a series of testing and validation phases, we established a protocol for FFPE-NT genotyping with results comparable with blood genotyping. The median call rate of FFPE-NT samples in the validation phase was 99.85% (range 98.26%-99.94%) and median concordance with matching blood samples was 99.79% (range 98.85%-99.9%). We also demonstrated that a rare pathogenic PALB2 genetic variant predisposing to cancer can be correctly identified in FFPE-NT samples. We further imputed the FFPE-NT genotype data and calculated the FFPE-NT genome-wide PRS in 3 diseases and 4 disease risk variables. In all cases, FFPE-NT and matching blood PRS were highly concordant (all Pearson's r > 0.95). The ability to precisely genotype FFPE-NT on a genome-wide array enables translational genomics applications of archived FFPE-NT samples with the possibility to link to corresponding phenotypes and longitudinal health data.

Formalin-Fixed Paraffin-Embedded Proteomics of Malignant Mesothelioma and New Candidate Biomarkers Thioredoxin and Superoxide Dismutase 2 for Immunohistochemistry.

The pathogenesis of malignant mesothelioma (MM) has been extensively investigated, focusing on stress derived from reactive oxygen species. We aimed to identify diagnostic biomarkers of MM by analyzing proteins in formalin-fixed paraffin-embedded specimens using liquid chromatography-mass spectrometry. We extracted proteins from formalin-fixed paraffin-embedded sections of MM tissues (n = 7) and compared their profiles with those of benign mesothelial tissues (n = 4) and alveolar tissue (n = 1). Proteomic data were statistically assessed and profiled using principal component analysis. We were successful in the classification of MM and healthy tissue. The levels of superoxide dismutase 2 (SOD2), an enzyme that converts superoxide anion into oxygen and hydrogen peroxide, and thioredoxin (TXN), which plays a crucial role in reducing disulfide bonds in proteins, primarily contributed to the classification. Other redox-related proteins, such as pyruvate dehydrogenase subunit X, and ceruloplasmin also contributed to the classification. Protein-protein interaction analysis demonstrated that these proteins play essential roles in MM pathogenesis. Immunohistochemistry revealed that TXN levels were significantly lower, whereas SOD2 levels were significantly higher in MM and lung cancer tissues than in controls. Proteomic profiling suggested that MM tissues experienced increased exposure to hydrogen peroxide and other reactive oxygen species. Combining immunohistochemistry for TXN and SOD2 allows for differentiation among MM, lung cancer, and control tissues; hence, TXN and SOD2 may be promising MM biomarkers and therapeutic targets.

OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

Spatial transcriptomics and the anatomical pathologist: Molecular meets morphology.

In recent years anatomical pathology has been revolutionised by the incorporation of molecular findings into routine diagnostic practice, and in some diseases the presence of specific molecular alterations are now essential for diagnosis. Spatial transcriptomics describes a group of technologies that provide up to transcriptome-wide expression profiling while preserving the spatial origin of the data, with many of these technologies able to provide these data using a single tissue section. Spatial transcriptomics allows expression profiling of highly specific areas within a tissue section potentially to subcellular resolution, and allows correlation of expression data with morphology, tissue type and location relative to other structures. While largely still research laboratory-based, several spatial transcriptomics methods have now achieved compatibility with formalin-fixed paraffin-embedded tissue (FFPE), allowing their use in diagnostic tissue samples, and with further development potentially leading to their incorporation in routine anatomical pathology practice. This mini review provides an overview of spatial transcriptomics methods, with an emphasis on platforms compatible with FFPE tissue, approaches to assess the data and potential applications in anatomical pathology practice.

Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays.

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.

A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples.

RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.

Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.

Targeted Quantitative Mass Spectrometry Analysis of Protein Biomarkers From Previously Stained Single Formalin-Fixed Paraffin-Embedded Tissue Sections.

Formalin-fixed, paraffin-embedded tissues represent a majority of all biopsy specimens commonly analyzed by histologic or immunohistochemical staining with adhesive coverslips attached. Mass spectrometry (MS) has recently been used to precisely quantify proteins in samples consisting of multiple unstained formalin-fixed, paraffin-embedded sections. Here, we report an MS method to analyze proteins from a single coverslipped 4-µm section previously stained with hematoxylin and eosin, Masson trichrome, or 3,3'-diaminobenzidine-based immunohistochemical staining. We analyzed serial unstained and stained sections from non-small cell lung cancer specimens for proteins of varying abundance (PD-L1, RB1, CD73, and HLA-DRA). Coverslips were removed by soaking in xylene, and after tryptic digestion, peptides were analyzed by targeted high-resolution liquid chromatography with tandem MS with stable isotope-labeled peptide standards. The low-abundance proteins RB1 and PD-L1 were quantified in 31 and 35 of 50 total sections analyzed, respectively, whereas higher abundance CD73 and HLA-DRA were quantified in 49 and 50 sections, respectively. The inclusion of targeted ß-actin measurement enabled normalization in samples where residual stain interfered with bulk protein quantitation by colorimetric assay. Measurement coefficient of variations for 5 replicate slides (hematoxylin and eosin stained vs unstained) from each block ranged from 3% to 18% for PD-L1, from 1% to 36% for RB1, 3% to 21% for CD73, and 4% to 29% for HLA-DRA. Collectively, these results demonstrate that targeted MS protein quantification can add a valuable data layer to clinical tissue specimens after assessment for standard pathology end points.

Panoramic Tissue Examination That Integrates 3-Dimensional Pathology Imaging and Gene Mutation: Potential Utility in Non-Small Cell Lung Cancer.

Novel therapeutics have significantly improved the survival and quality of life of patients with malignancies in this century. Versatile precision diagnostic data were used to formulate personalized therapeutic strategies for patients. However, the cost of extensive information depends on the consumption of the specimen, raising the challenges of effective specimen utilization, particularly in small biopsies. In this study, we proposed a tissue-processing cascaded protocol that obtains 3-dimensional (3D) protein expression spatial distribution and mutation analysis from an identical specimen. In order to reuse the thick section tissue evaluated after the 3D pathology technique, we designed a novel high-flatness agarose-embedded method that could improve tissue utilization rate by 1.52 fold, whereas it reduced the tissue-processing time by 80% compared with the traditional paraffin-embedding method. In animal studies, we demonstrated that the protocol would not affect the results of DNA mutation analysis. Furthermore, we explored the utility of this approach in non-small cell lung cancer because it is a compelling application for this innovation. We used 35 cases including 7 cases of biopsy specimens of non-small cell lung cancer to simulate future clinical application. The cascaded protocol consumed 150-µm thickness of formalin-fixed, paraffin-embedded specimens, providing 3D histologic and immunohistochemical information approximately 38 times that of the current paraffin-embedding protocol, and 3 rounds of DNA mutation analysis, offering both essential guidance for routine diagnostic evaluation and advanced information for precision medicine. Our designed integrated workflow provides an alternative way for pathological examination and paves the way for multidimensional tumor tissue assessment.

Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia.

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p < 0.001) and RNA (fail to qualify ratio = 1070:280 days, p < 0.001). After improvement of pre-analytical factors, the qualification rate increased for hospitals A and E from 41.5% to 70.5% and 62.5% to 86%, respectively. This is the first report to elucidate the pre-analytical laboratory procedures of main Southeast Asian oncology hospitals. An external quality assessment program may improve factors associated with tumor FFPE specimen quality.

Routine Workflow of Spatial Proteomics on Micro-formalin-Fixed Paraffin-Embedded Tissues.

In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed paraffin-embedded (FFPE) tissues are an important material in spatial proteomics, in which fixed tissues are excised using laser capture microdissection (LCM), followed by protein identification with mass spectrometry. For a satisfied spatial proteomics upon FFPE tissues, the excision area is expected to be as small as possible, and the identified proteins are countered upon as much as possible. For a general laboratory for spatial proteomics, a routine workflow is required, not relying on any special device, and is easily operating. In view of these challenges in technology, we initiated a technology evaluation throughout the entire procedure of proteomic analysis with micro-FFPE tissues. In contrast to the protocols reported previously, several innovations in technology were proposed and conducted, such as removal of destaining, decross-linking with "hang-down", solution simplification for peptide generation and balancing to excision area, and capture rate of micro-FFPE tissues. After optimization of all the necessary steps, a routine workflow was established, in which the minimized area for protein identification was 0.002 mm2, while the excision area for a consistent proteomic analysis was 0.05 mm2. Using the developed workflow and collecting the micro-FFPE tissues continuously, for the first time, a spatial proteomic atlas of mouse brain was preliminarily constructed, which exhibited the typical characteristics of spatial-dependent protein abundance and functional enrichment.

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.

Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.

INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times. AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant. EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

OMIP-088: Twenty-target imaging mass cytometry panel for major cell populations in mouse formalin fixed paraffin embedded liver.

The purpose of this 20-target imaging mass cytometry (IMC) panel is to identify the main cell types in formalin fixed paraffin embedded (FFPE) mouse liver tissue with the Hyperion™ mass cytometer from Standard BioTools (formerly Fluidigm). The antibody panel includes markers to identify hepatocytes (E-cadherin, HNF4α (hepatocyte nuclear factor 4 alpha), Arginase-1), liver sinusoidal endothelial cells (LSECs; CD206), Kupffer cells (F4/80, CD206), neutrophils (Ly6G, CD11b), bone marrow derived myeloid cells (BMDMs; CD11b), cholangiocytes (E-cadherin high), endothelial cells (CD31, α-SMA), plasmacytoid dendritic cells (CD317), B cells (CD19), T cells (CD3e, CD4, CD8a), NK cells (CD161) as well markers of cell activation (CD44, CD74), proliferation (Ki-67) and to aid in cell segmentation (Pan-Actin, E-cadherin, histone H3). The panel has been tested in other mouse tissues, namely the spleen, colon and lung, and therefore is likely to work across various mouse FFPE samples of interest. It has not been tested using human samples, frozen samples or in suspension mass cytometry because FFPE treatment profoundly changes epitope conformation. In summary, this panel is a powerful tool for pre-clinical research to determine cellular abundance and spatial distribution within mouse tissues and serves as a scaffold, to which more targets can be added for project specific requirements.