ATP-Coated Dual-Functionalized Titanium(IV) IMAC Material for Simultaneous Enrichment and Separation of Glycopeptides and Phosphopeptides

Abstract

Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which play an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. In this study, we developed a hydrophilicity-enhanced bifunctional Ti-IMAC (IMAC: immobilized metal affinity chromatography) material with grafted adenosine triphosphate (denoted as epoxy-ATP-Ti 4+ ) to enable simultaneous enrichment and separation of common N-glycopeptides, phosphopeptides, and M6P glycopeptides from tissue/cells. The enrichment was achieved through a dual-mode mechanism based on the electrostatic and hydrophilic properties of the material. The epoxy-ATP-Ti 4+ IMAC material was prepared from epoxy-functionalized silica particles via a convenient two-step process. The ATP molecule provided strong and active phosphate sites for binding phosphopeptides in the conventional IMAC mode and also contributed significantly to the hydrophilicity, which permitted the enrichment of glycopeptides via hydrophilic interaction chromatography. The two modes could be implemented simultaneously, allowing glycopeptides and phosphopeptides to be collected sequentially in a single experiment from the same sample. In addition to standard protein samples, the material was further applied to glycopeptide and phosphopeptide enrichment and characterization from HeLa cell digests and mouse lung tissue samples. In total, 2928 glycopeptides and 3051 phosphopeptides were identified from the mouse lung tissue sample, supporting the utility of this material for large-scale PTM analysis of complex biological samples. Overall, the newly developed epoxy-ATP-Ti 4+ IMAC material and associated fractionation method enable simple and effective enrichment and separation of glycopeptides and phosphopeptides, offering a useful tool to study potential crosstalk between these two important PTMs in biological systems. The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029775.

An efficient strategy with a synergistic effect of hydrophilic and electrostatic interactions for simultaneous enrichment of N- and O-glycopeptides

“…Since the enrichment of glycopeptides using PDA–ADE@KCP involves the synergistic effect of hydrophilic and electrostatic interactions, the effect of synergism on the glycopeptide enrichment results was first investigated. According to existing research, in the HILIC mode, the elution conditions of 0.1% TFA followed by NH 4 HCO 3 were used in previous research, 54,55 the former can effectively elute glycopeptides captured via hydrophilic interactions, while the latter can effectively elute glycopeptides captured via electrostatic interactions. Therefore, in order to determine whether the synergistic effect could actually improve glycopeptide identification, the results after eluting glycopeptides using 0.1% TFA and the composition of 0.1% TFA and 25 mM NH 4 HCO 3 were compared, respectively.…”

Cotton Ti-IMAC: Developing Phosphorylated Cotton as a Novel Platform for Phosphopeptide Enrichment

“…LC-MS/MS raw data files were processed using MaxQuant software (Version 1.6.7.0) following previously described settings. , In brief, the UniProt Homo sapiens reviewed database (August 2020, 20311 entries) was used as the protein database. Mass tolerance for precursor ions and fragment ions was specified as 4.5 and 20 ppm, respectively.…”

“…Given the numerous merits of the material, it is anticipated to serve as a widely applicable and robust platform for large-scale phosphopeptide enrichment, which can provide a deeper understanding of the phosphoproteome in any complex biological or clinical samples. By taking advantage of the intrinsic hydrophilicity of cotton fiber, future implementation of Cotton Ti-IMAC could also be extended to enrich other PTMs simultaneously with phosphorylation. ,, …”

“…The samples were analyzed by MALDI-MS and LC-MS/MS using previously described instrument parameters . Briefly, for MALDI-MS, standard protein samples were reconstituted in 0.1% FA and spotted on a plate with a DHB matrix (25 mg/mL in 50% ACN/1% H 3 PO 4 (v/v)).…”

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics

“…Furthermore, a new material, epoxy-adenosine triphosphate (ATP)-Ti(IV)-IMAC, has been leveraged for the concurrent enrichment of phosphopeptides and N-glycopeptides, capitalizing on the high hydrophilicity of ATP and its metal ion chelation ability(29). The second strategy combines two methodologies for sequential enrichment of the two PTMs(30).…”