Enrichment of Phosphopeptides by Fe³⁺-Immobilized Magnetic Nanoparticles for Phosphoproteome Analysis of the Plasma Membrane of Mouse Liver

Abstract

Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe3+-immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein alpha-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDI-TOFMS analysis. The lowest detectable concentration is 5 x 10(-11) M for 100 microL of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

“…Scansite datasets used for comparative analysis were retrieved from largescale phosphoproteomics studies in human liver (35), primary human multiple myeloma cells (36), HeLa nuclear protein (37), mouse liver (38), zebrafish embryo (39), Trypanosoma brucei (40), and S. cerevisiae (41). For those phosphorylation sites that we were unable to assign to a kinase recognition motif when using the Scansite algorithm under low stringency, the Motif-X algorithm was employed to identify phosphorylation motifs (42).…”

Ti4+‐Immobilized Magnetic Composite Microspheres for Highly Selective Enrichment of Phosphopeptides

“…[23][24][25][26] Along this line, the application of magnetic composite microspheres to proteomics research has also garnered much attention. [27][28][29][30][31][32] Post-translational modifi cations (PTMs) are chemical modifi cations of a protein performed in order to broaden its biological functionality. Reversible protein phosphorylation, as one of the most important protein PTMs, plays a vital role in regulating many complex biological processes, such as cellular growth and division, and signaling transduction.…”

Recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics

“…For the former strategy, various base materials have been adopted considering their own properties: nanozeolites [83] with excellent modifiability of surface hydroxyl groups, mesoporous molecular sieves MCM-41 [84,85], whose surface has been covered by a large amount of silicate hydroxyl groups, and magnetic nanoparticles [86], readily to be separated by an external magnet. For the latter strategy, metal oxide nanoparticles could be directly used as the solid-phase extraction adsorbents to capture phosphopeptides from bulk volume of sample solutions [87,88], or packed into cartridge or pipette tip as chromatography materials to bind phosphopeptides while the sample solution flows through it [89,90].…”