Layered electrophoretic transfer – A method for pre‐analytic processing of histological sections

Abstract

Current technologies for measuring protein expression across a tissue section are based on mass spectrometry or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico- chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed Layered Electrophoretic Transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: 1) A reduced complexity of the tissue proteome for subsequent analysis; 2) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or mass spectrometry for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.

The Use of Electrophoresis for the Study of Saliva Involvement in Ingestive Behavior

“…The stability of proteins is achieved through cross-linking, with multiple reports showing that protein modifications, such as phosphorylation are maintained and can be determined years later by immunohistochemistry [104]. As such the FFPE tissues have the potential to constitute a valuable source of samples for proteome analysis using electrophoresis [105,106]. Protein extraction from FFPE tissue samples allows the generation of protein profiles by high-throughput techniques, providing an improvement over other traditional methods such as immunohistochemistry [107,108].…”

In Situ Proteomic Analysis of Histological Sections

“…Layered expression scanning (LES) is a technology codeveloped by the National Cancer Institute and 20/20 GeneSystems, Inc. (http://www.2020gene.com) [16][17][18][19][78][79][80]. Based on the transfer of biomolecules from a tissue section through a stack of membrane layers, LES provides an alternative to IHC and IMS for multiplex in situ analysis [16][17][18][19][78][79][80].…”

“…Based on the transfer of biomolecules from a tissue section through a stack of membrane layers, LES provides an alternative to IHC and IMS for multiplex in situ analysis [16][17][18][19][78][79][80]. One of the key aspects of the technology is the ability to maintain the two-dimensional architecture of the tissue sample throughout the process (Fig.…”

“…Antibodies captured by peptides coated on P-Film membrane of the first technologies to perform in situ protein separation on a tissue specimen [19]. In LET, proteins are electrophoresed from an intact tissue section through either one or multiple polyacrylamide gel layers.…”

“…Despite some limitations, MALDI-IMS offers the first attempt to directly scan a tissue section and obtain a small molecule read-out. In addition, there are several new in situ analysis techniques that are under development, including the layered expression scanning (LES) suite of technologies [16][17][18][19]. The present review summarizes these proteomic technologies and discusses their applications in the field of molecular pathology.…”