Analysis of Cell Surface Proteome Changes via Label-free, Quantitative Mass Spectrometry

Ralph Schiess,Lukas N Mueller,Alexander Schmidt,Markus Mueller,Bernd Wollscheid,Ruedi Aebersold

doi:10.1074/mcp.m800172-mcp200

Abstract

We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. The method is based on the label-free quantification of peptide patterns acquired by high mass accuracy mass spectrometry using new software tools and the cell surface capturing technology that selectively enriches glycopeptides exposed to the cell exterior. The method was applied to monitor dynamic protein changes in the cell surface glycoproteome of Drosophila melanogaster cells. The results led to the construction of a cell surface glycoprotein atlas consisting of 202 cell surface glycoproteins of D. melanogaster Kc167 cells and indicated relative quantitative changes of cell surface glycoproteins in four different cellular states. Furthermore we specifically investigated cell surface proteome changes upon prolonged insulin stimulation. The data revealed insulin-dependent cell surface glycoprotein dynamics, including insulin receptor internalization, and linked these changes to intracellular signaling networks.

Highlights

We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes
To facilitate the deep and specific analysis of cell surface proteins we recently developed a method for the selective identification of cell surface glycoproteins, the cell surface capturing (CSC) method
We approached this by combining the selective enrichment of cell surface glycoproteins with a label-free, quantitative proteomics method that provides the sample throughput required to analyze multiple samples

Summary

Introduction

We present a mass spectrometry-based strategy for the specific detection and quantification of cell surface proteome changes. As an alternative to the analysis of total cell or tissue extracts that leads to the identification and, if suitable quantification strategies are applied [4], to the quantification of a fraction of the proteins present in the sample, analysis of specific subproteomes that are enriched for proteins of particular types has been suggested [5] Implementations of this concept so far include the selective isolation and subsequent analysis of cysteine-containing peptides [6], phosphorylated peptides [7], N-glycosylated peptides [8], the set of N-terminal peptides [9], and specific subcellular fractions and organelles [10, 11].

Objectives

Results

Conclusion