PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells

Abstract

Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

“…Proteins from the cell surface are isolated from detergent extracts by affinity chromatography with immobilized avidin and identified with MS [21]. Bledi et al [38] have applied proteases to intact cells. The resulting peptide fragments were further analyzed by LC followed by MS/MS.…”

Aptamer Directly Evolved from Live Cells Recognizes Membrane Bound Immunoglobin Heavy Mu Chain in Burkitt's Lymphoma Cells

“…There are several approaches, such as genetic and proteomics methodologies for the identification of membrane proteins (5,6). These methods are mainly designed to identify differences in protein expression patterns of cancer cell samples compared with healthy ones.…”

Moving Receptor Redirected Adoptive Cell Therapy Toward Fine Tuning of Antitumor Responses

“…Initial studies relied on the biochemical fractionation of the membrane and posterior isolation of proteins [48,49], but this method has disadvantages like contamination from other cellular compartments. In other approach, intact cells are "shaved" by proteases and the fragments are purified and analyzed by mass spectrometry, bypassing the membrane fractionation step [50,51]. In vivo analysis using phage libraries are being tested [52] and such approaches can be International Reviews of Immunology Int Rev Immunol Downloaded from informahealthcare.com by Akademiska Sjukhuset on 10/14/14…”