Abstract
The activity of membrane proteins are critically controlled by post-translational modification (PTM), such as glycosylation in the extracellular domain that functions as signal receiver, while the phosphorylation occurring at the intracellular domain initiates the signaling transduction pathway. However, it is still a great challenge to analyze them due to the bottlenecks of highly hydrophobic nature of membrane proteins, low stoichiometry on modification, heterogeneous PTM pattern and ion suppression effect from unmodified peptides of high abundance. To facilitate concomitant analysis of membrane proteome and their PTM sites, we proposed a quantitation platform by integration of deglycosylation-based spectra enrichment, IMAC enrichment and iTRAQ labeling strategies. The performance of spectra enrichment was first demonstrated the glycopeptides can be identified based on two criteria: 1) consensus sequence Asn-Xxx-Ser/Thr with mass shift of 1 Da; 2) iTRAQ ratio with 2-fold enhancement. Further incorporation of 18O-labelig in the sequential digestion steps, the iTRAQ-based signal enhancement with 18O-labeled deglycosylation achieved 100% confidence for accessing glycosylation site occupancies. This strategy also offers advantage to discriminate alterations at either the protein expression level or the modification extent on glycosylation and phosphorylation under different biological states. We integrated this strategy with IMAC purification for quantitative analysis on the B cell lymphoma in response to cytokine stimulation. The differential expression of glycosylation sites and phosphorylation sites revealed that many proteins involved in the immune response and receptor signaling pathway from B cell lymphoma in response to IL-2/IL5 stimulation. We expected that this spectra enrichment strategy may provide a new method for concomitant analysis of N-glycosylated and phosphorylated membrane proteome. Next, a new type of hybrid biomaterial, namely a boronicacid-decoratedlectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment was developed and applied on the level of whole cell lysates for glycoproteomic analysis. Three different types of BAD-lectin@MNPs exhibited excellent specificities for glycopeptide enrichment. Furthermore, the precursor ion discovery (PID) mode from Q-TOF-MS was used to filter glycopeptide spectra, the enrichment selectivity was observed that each BAD-ConA@MNP, BAD-AAL@MNP and BAD-SNA@MNP contain the majority of characteristic fragments of their corresponding recognition glycan. With the demonstrated enrichment selectivity and enhanced extraction efficiency, the reported BA-Lectin@MNP-based mass spectrometric method provides a glycan-targeting tool to facilitate the in-depth analysis of glycoproteome.