Project description:Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.
Project description:Co-polymer 1,2-epoxy-5-hexene and divinyl benzene with terminal oxirane group is synthesized for the first time. High density of hydrophilic groups (-COOH, -OH) enhances hydrophilicity through facile functionalization with Diethylene Triamine (DETA) and chloroacetic acid. COOH-functionalized mesoporous polymeric beads provide high surface area and porosity, superior hydrophilicity, solvent resistance and good bio-capability for enriching N-Linked glycopeptides. Sensitivity, selectivity, reusability and batch to batch reproducibility are tested using model glycoproteins (HRP, IgG and avidin) and analyzed by MALDI-TOF-MS. Furthermore, N-glycosylation sites in N-glycopeptides of characteristic glycoproteins are identified from digested human serum.
Project description:1. Profiling of sialylated glycopeptides from rEPO was performed via LC–HRMS 2. LC–HRMS methods for analyzing sialylated glycopeptide in urine samples were developed 3. The method was validated and applied to detection of rEPO biosimilars in urine
Project description:We employed the newly available IMPa O-glycoprotease from Pseudomonas aeruginosa for O-glycoproteomics analysis of cultured cells and tissues. The glycopeptides were extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by EThcD, which allowed for a site-specific and global analysis of O-glycans, especially those sialylated and the Tn antigen. Through wild-type or SimpleCell HEK293 cells, we present two approaches, one for the analysis of total O-glycoproteome and the other for the Tn glycoproteome, and showed that IMPa and EThcD are necessary for confident localization of O-glycans on glycopeptides. We applied this method to study the O-glycoproteome of mouse brain, which revealed the high frequency of sialylated O-glycans and the Tn antigen on brain glycoproteins.
Project description:Simultaneous enrichment and fractionation of diverse proteins/peptides possessing different post-translational modifications (PTMs) from the same biological samples is highly desirable to reduce sample consumption, avoid complicated sample processing, and enable studies of potential crosstalks between different PTMs. In this work, we report a new approach to enable simultaneous enrichment and separation of glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides by using a dual-functional Ti(IV)-IMAC material. Moreover, we also made the separation of neutral and sialyl glycopeptides and mono- and multi-phosphopeptides possible by performing different elution processes according to the differences in their electrostatic or hydrophilic properties. These separations are effective and efficient to eliminate the signal suppression from neutral glycopeptides for sialyl glycopeptide detection, allowing separation of mono-phosphopeptides from multi-phosphopeptides, as well as detection of M6P glycopeptides that are free from the abovementioned modifications. This new strategy significantly improves the coverage and identification numbers of glycopeptides, phosphopeptides, and M6P glycopeptides by 1.9, 2.3, and 4.3-fold compared with the conventional method, respectively. This is the first report on simultaneous enrichment and separation of neutral and sialyl glycopeptides, mono- and multi-phosphopeptides, and M6P glycopeptides via dual-functional Ti(IV)- IMAC, revealing novel insights into potential crosstalk among these important PTMs.
Project description:Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Correlation of the intact glycoform to the protein attachment site is a critical step to assign functional roles to specific glycoproteins. Isotope targeted glycoproteomics (IsoTaG) is a mass-independent mass spectrometry method to characterize intact, metabolically labeled glycopeptides from complex proteomes. IsoTaG was applied to conditioned media from PC-3 cells labeled with alkynyl or azido sugars to reveal the sialylated glycoproteome. Analysis on an Orbitrap Fusion Tribrid mass spectrometer resulted in characterization of 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycoforms across 126 Nand 576 O-glycopeptides. IsoTaG is an effective platform for identification of intact glycopeptides labeled by alkynyl or azido-glycans and will facilitate further studies on the role of the glycoproteome in biology.