Synthesis Of Fe~Ⅲ-TA Films-functionalized Magnetic Silica Microspheres For The Enrichment Of Low-abundance Peptides And Proteins

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), endowed with simplicity, excellent mass accuracy, high resolution and sensitivity, has become an indispensible analytical tool for proteomic researches. Despite these advantages, it is still challenging for MALDI-TOF MS to trace low-abundance peptides/proteins due to their extremely low concentrations in real biological samples. Moreover, highly abundant peptides/proteins in the sample matrices or contaminants like salts and surfactants introduced in pretreatment procedures can also seriously interfere with the MS detection. Therefore, efficient enrichment of low-abundance peptides/proteins is critical prior to their MS detection.In this work, magnetic silica microspheres with a size distribution of 300 to 400 nm were firstly prepared through a sol-gel process. Then, novel FeⅢ-TA films-functionalized magnetic silica microspheres (Fe3O4@SiO2@FTA) were prepared via a facile deposition of one-step assembled FeⅢ-TA films on the surface of magnetic silica microspheres. The resulting microspheres were characterized by various techniques including SEM, TEM, FT-IR and SQUID VSM. Characterization results showed that the as-prepared microspheres are nearly spherical with uniform size and exhibit strong magnetic responsiveness and good dispersity in aqueous solution.We introduced low concentrations of standard peptide angiotensin Ⅱ, standard protein cytochrome C, tryptic digests of BSA and tryptic digests of protein spot from 2-DE gel and peptides in human urine to investigate the enrichment capability of the novel microspheres. After enrichment, signal intensities and S/N ratios of spectra of proteins and peptides are sharply enhanced, indicating the material possess decent enrichment effect towards biological samples with different complexity. The enrichment process was very fast (only 5 min) and the peptides/proteins-magnetic microspheres conjugates were readily separated by an external magnet. Moreover, the obtained conjugates can be directly analyzed by MALDI-TOF MS without any elution procedure. After repeated verification, the material exhibits a stable enrichment performance and delivers a good data reproducibility, indicating Fe3O4@SiO2@FTA microsphere a promising material for the efficient and fast enrichment of low-abundance peptides and proteins.