Journal: British Journal of Cancer

Article Title: Proteomic and metabonomic biomarkers for hepatocellular carcinoma: a comprehensive review

doi: 10.1038/bjc.2015.38

Figure Lengend Snippet: Summary of proteomic studies that were reviewed

Article Snippet: , , Tissue , ↑ HCC:CON , 100% of HCCs , 6 HCC, 6 non-malignant adjacent tissue , HBV , — , 2.1 † , 2DE-coupled MALDI–TOF–MS , Matos et al , 2009 .

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Protease Inhibitor, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Study workflow. H&E slides were annotated by a breast pathologist with regions defined as DCIS (blue) or IBC (red or orange). On a subsequent tissue section, slides were prepared for collagenase digestion to target the extracellular matrix (ECM). Mass spectrometry imaging (MSI) was performed with matrix-assisted laser desorption/ionization–quadrupole time-of-flight (MALDI-QTOF) imaging. Four samples were annotated per pathological lesion type and underwent the collagenase MSI workflow with high-resolution imaging at an individual lesion level. From the remaining eighteen-sample subset, specific slides were selected for further proteomic analysis multiplexing either tryptic or elastase digestion followed by mass spectrometry imaging. This schema was created in Biorender.com.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Mass Spectrometry, Imaging, Multiplexing

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Spatial mapping of the extracellular proteome defines DCIS histopathology. ( A ) The eighteen-sample cohort underwent the workflow depicted, beginning with pathological annotation followed by extracellular matrix (ECM)-targeted mass spectrometry imaging and ECM peptide identification. ( B ) Spectra from pathologist-defined lesions with DCIS shown in blue and IDC shown in red demonstrate different relative peak intensity profiles. R. int. denotes the normalized relative intensity of peaks computed in mMass ® . ( C ) Hematoxylin and eosin-stained image of a mixed DCIS-IDC specimen demonstrates DCIS (blue) and IDC pathology (red). ( D ) Spatial segmentation analysis was used to define five main proteomic clusters. Cluster 1 (dark blue) annotates to adipocyte regions; Cluster 2 (green) defines borders between adipocyte and stroma; Cluster 3 (pink) localizes to stroma that includes DCIS lesions; Cluster 4 (blue) is localized to stroma and adipocytes primarily between tumor and adjacent tissue; Cluster 5 (yellow) annotates to the cancer region with diminishing detection distant from the tumor. ( E ) Pie chart depicting the proportion of peptide sequences identified from select protein classifications. Collagen fraction is further divided into collagen structural categories. ( F ) Spatial heat maps of a ColIα2 peptide depicted in red show distinct localization to DCIS lesions and surrounding ductal regions compared to the filamin-C peptide, which borders ductal regions and localizes to IDC. INPPL1 denotes inositol polyphosphate phosphatase like 1. Images were normalized to an internal peptide standard. Putative identifications were made by matching imaging data to an ECM database. Numbers following identification indicate the amino acid positions within the entire protein sequence. ( G ) Extracellular matrix peptides distinguished between DCIS, IDC, and DCIS-IDC. Heatmap is the average peptide expression detected across tissue images. ( H ) Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) of pathological regions depicts distinct clustering of regions by specimen classifications of DCIS ( n = 9), mixed DCIS-IDC ( n = 6), and IDC ( n = 4). ( I ) Loadings plot from sPLS-DA depicts the top ten peptide peaks that discriminate between specimen types. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy. sPLS-DA and heat map analyses were performed with MetaboAnalyst 5.0.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Histopathology, Mass Spectrometry, Imaging, Staining, Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: DCIS specimens report distinct fibrillar collagen profiles to pathological regions. ( A ) A total of 43 extracellular matrix peptides were identified across tissue images distinguished between DCIS and IDC by an unpaired, two-tailed t -test ( p < 0.01). ( B ) A volcano plot of peaks identified via LC-MS/MS reports the most significantly differentially expressed peaks between DCIS and IDC pathologies. An absolute value fold change greater than 0.5 between DCIS and IDC with −log( p -value) greater than or equal to 1.5 is shown in orange if increased expression was found in IDC and blue if decreased expression was found in IDC. The volcano plot was created with VolcaNoseR. ( C ) Box-and-whiskers plots of fibrillar collagen sequences that are differentially expressed between DCIS ( n = 13) and IDC ( n = 10) lesions in eighteen samples by the Mann–Whitney test ( p < 0.05). ROC analyses of peaks adjacent to box-and-whiskers plots (AUROC > 0.75 and p < 0.05 by the Wilson/Brown t -test) are shown. Ox denotes oxidation, and HYP denotes hydroxylation of proline residues. ( D ) Location of the identified peptide within the protein sequence found to be differentially expressed by the Mann–Whitney test ( p < 0.05). ( E ) Spatial heatmaps of MALDI-QTOF imaging of 1084.498 m/z and 1458.700 m/z from two representative samples. Black annotations encircle IDC regions, while white annotations delineate DCIS regions. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Expressing, MANN-WHITNEY, Sequencing, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Extracellular Microenvironment Alterations in Ductal Carcinoma In Situ and Invasive Breast Cancer Pathologies by Multiplexed Spatial Proteomics

doi: 10.3390/ijms25126748

Figure Lengend Snippet: Serial enzymatic digest reveals pathology-specific proteomes and proteomic field cancerization. ( A ) Workflow for serial enzymatic digestion depicted with cellular localization from LC-MS/MS proteomic hits from each enzymatic digestion shown. Tissue was digested by collagenase to define stroma composition, trypsin to capture cellular features and additional extracellular composition, and elastase to target elastin. ( B ) Pathologist-defined lesions annotated by architectural pattern and distance to the invasive cancer site. ( C ) Segmentation analysis from 53 peptides derived from stroma. The tumor (yellow) and adjacent tissue (blue) represent distinct clusters with stromal composition extruding from the tumor (green). ( D ) DCIS lesions show distinct stromal signatures dependent on distance from IDC. ( E ) Spatial heatmaps of 3 collagenase peptide peaks (1291.664 m/z, 1458.701 m/z, 1588.781 m/z) depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. ( F ) Segmentation from serial tryptic digestion highlights the invasive cancer field (pink) and normal adjacent tissue (blue). Potential margins and punctate extensions form a unique cluster (green). ( G ) Differential expression detected by tryptic peptides based on location relative to IDC. ( H ) Spatial heatmaps of 3 tryptic peptide peaks (958.566 m/z, 1797.841 m/z, 1550.809 m/z) depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. ( I ) Segmentation analysis of peptides derived from elastase digestion. The tumor field (yellow and green) extends further into the normal adjacent tissue (blue and purple) compared to tryptic segmentation profiles. ( J ) DCIS lesions show differential signatures derived from 393 peptides produced by elastase digestion dependent on distance from IDC. ( K ) Spatial heatmaps of 3 elastase peptide peaks (906.472 m/z, 854.462 m/z, 1240.669 m/z) identified from an elastase-digested peptide library depict discrete localization to IDC, DCIS, and surrounding normal adjacent tissue. Peptide sequences depicted within protein schemas. Ppm calculations between MALDI-QTOF imaging and LC-MS/MS were within 5 mass accuracy.

Article Snippet: COLase3, elastase, or trypsin was applied to slides using a M3 or M5 TM-Sprayer Tissue MALDI Sample Preparation System (HTX Technologies, LLC, Chapel Hill, NC, USA) with the following settings: 40 °C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes.

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Quantitative Proteomics, Produced, Imaging