Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

Abstract

Post-translational modifications (PTMs), mass spectrometry (MS), liquid chromatography (LC), tandem MS (MS/MS), phosphopeptide (p-peptide), label-free quantification (LFQ), stable isotope labelling by amino acids in cell culture (SILAC), Tandem Mass Tags (TMT) isobaric tags for relative and absolute quantification (iTRAQ), phospho-serine (pSer), threonine (pThr), tyrosine (pTyr), immobilized metal ion affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), acetonitrile (ACN), sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE), filter assisted sample preparation (FASP), solid phase extraction (SPE), high performance liquid chromatography (HPLC), reverse phase (RP), strong cation exchange (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), solution isoelectric focusing (sIEF), strong anion exchange (SAX), histidine (pHis), arginine (Arg),

Preparation of zirconium arsenate‐modified monolithic column for selective enrichment of phosphopeptides

“…Various metal ions (e.g. Fe 3+ , Ga 3+ , Ti 4+ , Hf 4+ , Ce 4+ , Sn 4+ , and Nb 5+ ) have been successfully employed for the selective enrichment of phosphopeptides in previous researches [6]. For comparison, TiAs‐IMAC monolithic column, FeAs‐IMAC monolithic column, and GaAs‐IMAC monolithic column were also prepared and applied to capture phosphopeptides from a tryptic digests of β‐casein and BSA mixture at the molar ratio of 1:100.…”

“…Traditionally, iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) were used as chelators for immobilizing metal ions. However, IDA and NTA have the disadvantages of limited specificity and metal ion loss [6]. In this case, novel chelators with higher specificity and chelating ability are desired.…”

“…However, the MS analysis of phosphopeptides is still a challenge due to the low abundance and low ionization efficiency of phosphopeptides as F I G U R E 1 Synthesis route for the preparation of ZrAs-IMAC monolithic column well as the serious interference from highly abundant nonphosphopeptides. Thus, selective enrichment of phosphopeptides from complex biological samples is essential prior to MS analysis [6].…”

“…Polymer monolith microextraction (PMME), as one kind of SPME using polymer monolith as the adsorbent, was proposed in our previous study [35] and has been successfully applied to the analysis of antidepressants, glycoproteins, and brassinosteroids [36][37][38]. Due to its good stability in a wide pH range, polymer monolith has a great potential in the IMAC-based phosphopeptide enrichment, since acidic solution and basic solution were commonly used for the extraction and elution, respectively [6]. In addition, the advantages of flexible synthesis, adjustable porosity, fast mass transport, and low column backpressure also make it an ideal substrate of IMAC adsorbent [39].…”

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies

“…Despite the robustness and continuous improvements of LFQ phosphoproteomics, the relatively low stoichiometry of some phosphoproteins limits the comprehensive analysis of protein phosphorylation [8]. Except for the prevention of phosphorylation and improved digestion efficiency in the proximity of the phosphorylated amino acid residues, the enrichment of samples with phosphopeptides is considered as the most critical step of phosphoproteomics [16][17][18]. Therefore, various phosphopeptide enrichment strategies have been developed.…”