Overcoming key technological challenges in using mass spectrometry for mapping cell surfaces in tissues

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.

Fig. 1.

Requirement for multiple replicate MS analysis of same sample for meaningful comparison of different samples. Two samples, tumor (T) and normal (N), were analyzed by standard MS methods to identify the total protein space (labeled a–z). As the number of replicates performed for each sample increases, the number of newly identified proteins in each sample decreases until 95% analytical completeness is reached. If the tumor and normal samples are compared (center column) before an adequate number of replicates have been analyzed, then false positives will prevail, e.g. the MS data in the first Venn diagram suggest that protein b is specific to the tumor sample, which is contrary to the Western blot analysis for this protein. Furthermore, when an additional MS replicate is performed for the normal sample, protein b is then detected by MS, confirming the findings of the Western blot. Only when multiple MS replicates are performed on both the tumor and normal samples can truly differentially expressed proteins be identified, e.g. proteins e and h. Note that only 25 of a possible 26 proteins (a–z) were identified, highlighting the fact that not all proteins can and will be detected by standard MS approaches. See text for more details.