Overcoming Key Technological Challenges in Using Mass Spectrometry for Mapping Cell Surfaces in Tissues

Noelle M Griffin,Jan E Schnitzer

doi:10.1074/mcp.r110.000935

Abstract

Plasma membranes form a critical biological interface between the inside of every cell and its external environment. Their roles in multiple key cellular functions make them important drug targets. However the protein composition of plasma membranes in general is poorly defined as the inherent properties of lipid embedded proteins, such as their hydrophobicity, low abundance, poor solubility and resistance to digestion and extraction makes them difficult to isolate, solubilize, and identify on a large scale by traditional mass spectrometry methods. Here we describe some of the significant advances that have occurred over the past ten years to address these challenges including: i) the development of new and improved membrane isolation techniques via either subfractionation or direct labeling and isolation of plasma membranes from cells and tissues; ii) modification of mass spectrometry methods to adapt to the hydrophobic nature of membrane proteins and peptides; iii) improvements to digestion protocols to compensate for the shortage of trypsin cleavage sites in lipid-embedded proteins, particularly multi-spanning proteins, and iv) the development of numerous bioinformatics tools which allow not only the identification and quantification of proteins, but also the prediction of membrane protein topology, membrane post-translational modifications and subcellular localization. This review emphasis the importance and difficulty of defining cells in proper patho- and physiological context to maintain the in vivo reality. We focus on how key technological challenges associated with the isolation and identification of cell surface proteins in tissues using mass spectrometry are being addressed in order to identify and quantify a comprehensive plasma membrane for drug and target discovery efforts.

Highlights

IntroductionElucidating the molecular topography of the cell surface in multiple tissues constitutes the first step in gaining a better fundamental understanding of functional differences across organ systems
There are key challenges associated with the identification and characterization of PM proteins, over the past 5 years, we as a field have made significant progress in overcoming some of the main obstacles associated with their proteomics analysis
We have developed a method (SIN) to control for variation between replicate samples so that real changes in protein abundance between biologically distinct samples can be detected [127]. This method allows the quantitative comparison of biologically distinct samples and should greatly facilitate the use of label-free quantitative proteomics approaches for differential protein expression analysis both in PM and other proteomes

Summary

Introduction

Elucidating the molecular topography of the cell surface in multiple tissues constitutes the first step in gaining a better fundamental understanding of functional differences across organ systems. We are making significant progress with characterizing ECs in vivo, such an approach needs to be applied to all in vivo cells as it is almost impossible to extrapolate in vitro findings into an in vivo situation. This is one challenge on which we need to focus because for biomarker and target discovery to move forward defining the in vivo reality becomes critical

Methods

Conclusion