Specific On-Plate Enrichment of Phosphorylated Peptides for Direct MALDI-TOF MS Analysis

Abstract

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

Photocatalytic Redox Reactions for In‐Source Peptide Fragmentation

“…[37] To remove impurity and decrease aggregation, the commercial P25 TiO 2 nanoparticles were heated (at 300 8C for 2 h) and then ground in a mortar (for 2 h). A stable suspension (100 mgml À1 ) was obtained by separating the nanoparticles in ethanol (89 %) and kept as the stock solution.…”

Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

“…Drinking milk was digested with trypsin by following the protocol described by Qiao et al 16 . A portion of 30 µL of drinking milk with 1.5% fat was diluted with 900 µL of 25 mM aqueous ammonium bicarbonate solution and centrifugated at 15 000 rpm for 20 minutes.…”

“…In order to enrich the phosphopeptides most of the new methods make use of the high affinity of the phosphoryl group to metals and metal oxides (e.g., on immobilized metal affinity chromatography (IMAC) [2][3][4][5] , metal oxide affinity chromatography (MOAC) 1,[6][7][8][9][10][11][12][13][14] , sequential immobilized metal affinity chromatography (SIMAC) 15 and direct on-target enrichment [16][17][18][19][20][21][22][23] ). Mass spectrometry (MS) is then commonly used to analyze the peptides.…”

Nanomaterial-Assisted Laser Desorption Ionization for Mass Spectrometry-Based Biomedical Analysis

“…The extracted molecules on the nanomaterials can then be directly ionized under laser irradiation and analyzed by MS. With this strategy, investigations, such as selective characterization of low-abundance molecules, MS-based enzyme immunoassay and MS-based DNA hybridization assay, were performed. As examples, TiO 2 , Al 2 O 3 and ZrO 2 were used to extract phosphorylated peptides from protein digests, and the enriched phosphopeptides were then analyzed directly by MALDI or NALDI-MS [66,[77][78][79][80][81]. Titania nanoparticles were also used as selective probes and matrices for the determination of catechins in tea using NALDI-MS [82].…”