Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes

Abstract

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics ofN-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.

Metabolomics, Transcriptomics and Functional Glycomics Reveals Bladder Cancer Cells Plasticity and Enhanced Aggressiveness Facing Hypoxia and Glucose Deprivation

“…Finally, cells predominantly expressing sialylated and fucosylated T antigen are markedly more invasive in vivo , without other major functional alterations. Building on available literature, we hypothesize that changing the glycosylation of membrane receptors may decisively impact on relevant downstream oncogenic pathways 1,92,93 . We are currently trying to identify common molecular grounds between hypoxic and glucose deprived cells and simple cell models that may account for these observations, which will be decisive to support future targeted therapeutic interventions.…”

Towards structure-focused glycoproteomics

“…In the surveyed studies, data were most frequently collected using data-dependent acquisition (DDA) by employing higher-energy collision dissociation (HCD-), stepped collision energy (SCE)-HCD-, electron-transfer dissociation (ETD)- and/or electron-transfer/higher-energy collision dissociation (EThcD-) MS/MS acquisition strategies (discussed below). Our survey also illustrated that targeted re-isolation and orthogonal fragmentation of glycopeptide ions are increasingly performed using diagnostic oxonium ions arising from HCD-MS/MS commonly referred to as product-dependent (pd) acquisition [ 60 , 66 , 67 , 71 ]. Data-independent acquisition (DIA), albeit less common, has also been used to profile mixtures of glycopeptides [ 57 , 72–76 ].…”

“…HILIC-based solid phase extraction (SPE) represents one of the least biased glycopeptide enrichment methods given the considerable (local) hydrophilicity shared by most N -glycopeptides [ 80 ], but may not quantitatively capture the less hydrophilic (shorter) O -glycopeptides and truncated N -glycopeptides [ 81–83 ]. To increase the glycoproteome coverage, some studies used multi-step enrichment protocols including sequential glycoprotein enrichment using Jacalin affinity chromatography followed by glycopeptide enrichment using HILIC [ 54 ] or employed sequential glycopeptide enrichment using HILIC and multi-lectin affinity (ConA, WGA and RCA 120) [ 39 ], while others applied parallel glycopeptide enrichment using ConA and mixed mode strong anion exchange-HILIC [ 60 ], or employed glycopeptide prefractionation using high-pH reversed-phase LC after the glycopeptide enrichment step [ 42 ].…”

“…Hybrid-type fragmentation methods that combine multiple dissociation methods include EThcD-MS/MS [ 210–213 ] widely used in glycoproteomics (see Table 1 for examples) and the less common AI-ETD-MS/MS method restricted to few specialist labs [ 41 , 60 ]. Fragmentation methods that employ multiple energy schemes to target more complete analyte dissociation most prominently the SCE-HCD-MS/MS method are also frequently used in large-scale glycopeptide profiling studies [ 37 , 51 , 56 , 214 , 215 ].…”

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics

“…Although this method has thus far been limited to characterization of formerly N-glycosylated peptides, it has been a valuable approach for generating cell surface glycoprotein maps of common mammalian cell lines and is used for a variety of applications (313)(314)(315)(316)(317)(318)(319)(320)(321)(322)(323)(324).…”