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. 2009 Apr;8(4):624-38.
doi: 10.1074/mcp.M800172-MCP200. Epub 2008 Nov 25.

Analysis of cell surface proteome changes via label-free, quantitative mass spectrometry

Ralph Schiess  1 , Lukas N Mueller, Alexander Schmidt, Markus Mueller, Bernd Wollscheid, Ruedi Aebersold
Affiliations

Analysis of cell surface proteome changes via label-free, quantitative mass spectrometry

Ralph Schiess et al. Mol Cell Proteomics. 2009 Apr.

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.

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Figures

F<sc>ig</sc>. 1.
Fig. 1.
Scheme of selective enrichment of cell surface glycoproteins. 1, the carbohydrate moieties of glycoproteins on the surface of intact, living cells were oxidized with sodium periodate. The thus chemically activated glycoproteins were then coupled to a biotin-containing linker molecule that does not damage or penetrate the cells. 2, the tagged cells were then lysed, cell debris and nuclei were removed by centrifugation, and a fraction enriched in membrane proteins was isolated from the lysate via ultracentrifugation. 3, upon digestion of the isolated membrane proteins, the tagged glycopeptides were affinity-purified using a streptavidin-covered solid support. 4, N-glycosites were then enzymatically released and analyzed by an LTQ-FT mass spectrometer. The obtained spectra were searched against the database BDGP5.2 using SEQUEST.
F<sc>ig</sc>. 2.
Fig. 2.
Cell surface LC-MS map of N-glycosites. A graphical representation of the LC-MS (RT versus m/z) feature map is shown. Only the identified N-glycosites are shown. Circled dots represent N-glycosites originating from the InR. The InR has 25 potential N-glycosylation sites of which 19 could be verified by the 44 N-glycosites described here. The data represented here can be found in supplemental Table S1.
F<sc>ig</sc>. 3.
Fig. 3.
Analysis of identified cell surface glycoproteins. a, GO cellular component analysis of the identified proteins. GO annotation for 126 proteins of the 202 glycoproteins identified was available. 86% belonged to the membrane, 5% belonged to the extracellular matrix, and 9% belonged to the cytoplasm. b, 183 of the 202 glycoproteins identified contain one or more TM domains as predicted by SOSUI (29). Furthermore 108 of the 126 GO-annotated proteins are membrane constituents.
F<sc>ig</sc>. 4.
Fig. 4.
Reproducibility of label-free quantification of the Kc167 cell surface glycoproteome. A scatterplot of LC-MS features of isolated N-glycosites using CSC and the respective squared Pearson correlation R2 are shown. a, the peptide peak areas of aligned MS1 features in the three replicate runs of the first experiment were plotted against each other. b, the mean peptide peak areas of aligned MS1 features in the three experiments were plotted against each other. Tech., technical; Exp., experimental.
F<sc>ig</sc>. 5.
Fig. 5.
Protein abundance changes on the cell surface upon differential perturbation. Glycoproteins from different perturbation experiments are shown in color according to their log ratio from green (4-fold down-regulated) to red (4-fold up-regulated). Glycoprotein ratios were built comparing each stimulated sample to a control. White fields indicate features that were not detected or quantified in the respective sample. Individual glycoprotein changes are listed in supplemental Table S6. PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor.
F<sc>ig</sc>. 6.
Fig. 6.
Quantitation of InR using isotope-coded protein labeling. a, MS spectra obtained over the whole elution time of VDLEHAN*NTESPVR, a formerly glycosylated peptide originating from the InR. The doubly charged species shows a lower signal for the light labeled peptide (d0; insulin-treated) than for the heavy labeled peptide (d4; control). b, relative abundance and the calculated d0:d4 ratio obtained using XPRESS software.
F<sc>ig</sc>. 7.
Fig. 7.
Protein abundance changes on the cell surface versus the whole cell membrane upon insulin stimulation. Protein ratios from insulin-treated versus non-treated cells obtained by CSC and whole membrane capturing are shown for integrin (a), InR (b), and baboon (c). d, protein log ratios obtained from both CSC (x axis) and whole membrane capturing (y axis) are plotted. Only protein ratios obtained in both experiments are shown. Proteins mentioned in the text are depicted as black dots. The corresponding abundance ratios of proteins quantified by both the CSC and the whole membrane capturing method are shown in bold in supplemental Tables S8 and S11.
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