A Shotgun Proteomic Method for the Identification of Membrane-Embedded Proteins and Peptides

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome

High-Throughput Microdissection for Next-Generation Sequencing

<div><p>Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, <i>in situ</i> hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.</p></div

xMD improves depth of NGS coverage in admixed cell cytospins.

<p>(A) Schematic image of the NGS workflow. xMD isolated targets were compared to manual macrodissection via NGS (B) Select variant evaluation of the 95% lymphoma (ST486)/5% lung carcinoma (A549) cell line specimens comparing manual macrodissection to xMD enrichment (C) Select variant evaluation of the 95% lymphoma (ST486)/5% melanoma (UACC.62) cell line specimens comparing manual macrodissection to xMD enrichment. ND = not detectable.</p

xMD application: Global epithelial microdissection.

<p>(A) a 1.25X digital image of the whole normal intestine specimen immunostained with cytokeratin AE1/AE3⁺ (B) a 1.25X digital image of the whole tissue following xMD, highlighting the degree of stained tissue procurement (C) a 1.25X digital image of the stained tissue bound to the xMD film (D-F) images of the before and after slide and film of the same specimen at higher (5x) magnification.</p

xMD applications: Nuclear and novel stain-based microdissection.

<p>(A) IHC stained p53⁺ nuclei from a section of metastatic colon carcinoma (B) IHC stained ER⁺ nuclei from a section of breast carcinoma (C) IHC stained GFP⁺ cells from a culture slide (D) EBER⁺ cells after EBV-EBER <i>in situ</i> hybridization (E) captured GMS⁺ <i>Aspergillus</i> fungal organisms.</p

xMD impact on NGS data quality for highly penetrant disease variants.

<p>xMD impact on NGS data quality for highly penetrant disease variants.</p