"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Proteins serve as the fundamental units by which the majority of cellular functions are carried out. Characterizing the structure and function of relevant proteins is an essential step to understand biological processes. Over the past decade, advances in mass spectrometry technology, analysis software, and databases have enabled the accurate detection and measurement of proteins at a proteome-wide scale¹. Mass spectrometry-based proteomics can be utilized in a diverse array of applications, from basic science analysis of biochemical pathways, to identification of novel drug targets in a translational setting, to diagnosis and monitoring of diseases in the clinic². When screening for novel drug targets, characterization of the cell surface proteome is particularly important, with over 65% of currently approved human drugs targeting cell surface proteins³. The field of cancer immunotherapy also wholly relies on cancer-specific cell surface antigens to target and specifically eliminate tumor cells⁴. Mass spectrometry-based proteomics can thus serve as a promising tool to identify new cell surface proteins toward therapeutic interventions.

However, there are several limitations when utilizing conventional proteomics methods to survey tumor cells for novel cell surface protein targets. A primary concern is that surface proteins make up a very small fraction of the total protein molecules in a cell. Therefore, fragments of these proteins are masked by a high abundance of intracellular proteins when performing mass spectrometry analysis of the whole-cell lysate⁵. This limitation makes it challenging to accurately characterize the cell surface proteome with a traditional proteomics workflow. To address this challenge, it is necessary to develop ways to enrich cell surface proteins out of the whole-cell lysate, prior to analysis on the mass spectrometer. One such method involves the oxidation and biotin labeling of glycosylated cell surface proteins in the intact cells, and subsequent enrichment of these biotinylated proteins from the lysate with a neutravidin pulldown, a process that has been termed "cell surface capture"⁶. Since ~85% of mammalian cell surface proteins are thought to be glycosylated⁷, this serves as an effective method of enriching the cell surface proteome out of the whole cell lysate. This paper describes a complete workflow, beginning with cultured cells, of cell surface biotin labeling, and subsequent sample preparation for mass spectrometry analysis (Figure 1). Over several replicates, this method provides robust coverage of the cell surface proteome of a particular sample. Utilizing this method to characterize the cell surface proteome of both tumor and healthy cells can facilitate the discovery of novel cell surface antigens to identify potential immunotherapeutic targets⁸.