Architectural design is essential to achieve ideal chemical and biological properties of nanomaterials. In this article, a novel route to fabricate high-quality magnetic composite microspheres composed of a high-magnetic-response magnetic colloid nanocrystal cluster (MCNC) core, a poly(methylacrylic acid) (PMAA) interim layer, and a Ti⁴⁺-immobilized poly(ethylene glycol methacrylate phosphate) (PEGMP) shell via two-step distillation–precipitation polymerization is presented. The unique as-synthesized MCNC@PMAA@PEGMP-Ti⁴⁺ composite microsphere is investigated for its applicability for selective enrichment of phosphopeptides from complex biological samples. The experiment results demonstrate that, by taking advantage of the pure phosphate–Ti⁴⁺ interface and high Ti⁴⁺ loading amount, the MCNC@PMAA@PEGMP-Ti⁴⁺ composite microsphere possesses remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/nonphosphopeptides (1:500). The extreme sensitivity, excellent recovery of phosphopeptides, and high magnetic susceptibility are also proven. These outstanding features demonstrate that the MCNC@PMAA@PEGMP-Ti⁴⁺ composite microspheres have great benefit for the pretreatment before mass spectrometric analysis of phosphopeptides. Furthermore, the performance of the approach in selective enrichment of phosphopeptides from drinking milk and human serum gives powerful evidence for its high selectivity and effectiveness in identifying the low-abundant phosphopeptides from complicated biological samples.

Phosphatidylcholines (PCs) are the most abundant phospholipids constituents of the cellular membrane, which exhibit a variety of biological functions. The distinct critical roles in cellular function make their analysis quite demanding. Although MALDI-MS is one of the most powerful tools for biomolecules identification, it is still limited to phospholipids studies. The ionization of phosphatidylcholines is insufficient, and signals of PCs are frequently confused and suppressed by matrix clusters occurring in the same mass range. As an alternative matrix, T-2-(3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene) malononitrile (DCTB) was introduced to overcome these problems in our study. Specifically, the signal intensity of phosphatidylcholines from soybean was enhanced more than several ten-folds using DCTB during positive ion MALDI-TOFMS. Peak overlaps caused by the wide distributions of series fatty acid residues from phospholipids were separated by introducing cesium cation. The occurred mass shift of 131.90 Da between [M+Cs]⁺ and [M+H]⁺ ("M" represents the molecular weight of the corresponding neutral hosphatidylcholine) was approved to be helpful in the assignments of ambiguous peaks of PCs from soybean. For real sample analysis, employing cesium chloride as an auxiliary reagent successfully facilitated the profiling and characterizing of PCs extracted from mouse lung and egg yolk followed by analysis with MALDI-TOFMS using DCTB as matrix.

The post translational modifications of histone variants are playing an important role in the structure of chro- matin, the regulation of gene activities and the diagnosis of diseases, and conducting in-depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ-Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂-terminal tails were also identified after the LC-MS/MS analysis of trypsin, Arg-C, Glu-C and chymotrypsin digests.

A core–satellite-structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica-coated ferrite “core” and numerous “satellites” of gold nanoparticles with lots of “anchors”. The anchor, 3-aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap-and-release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6 % separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N-glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1 %) were newly identified.

A new method based upon adding ammonium phosphate as a matrix additive to enhance the ionization efficiency of phosphopeptide in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was described. Furthermore, influences of different phosphate salts at various concentrations on the ionization efficiency of phosphopeptide were investigated systematically, finding that the signal intensity for phosphopeptide 48FQ[pS]EEQQQTEDELQDK63 digested from β-casein were 5 to 8 times increased in an optimized condition with 10 mmol/L ammonium monobasic phosphate or 3 to 4 times increased with 10 mmol/L ammonium dibasic phosphate as additive to matrix 2,5-dihydroxybenzoic acid, respectively. Compared with the most optimized matrix system that was currently reported for special ionization of phosphopeptides, the signal intensity of this phosphopeptide was also enhanced by twice when introducing 5 mmol/L ammonium dibasic phosphate into matrix 2,4,6-trihydroxyacetophenone. In addition, the mechanism was also discussed, assuming that the cooperation function of ammonium cation and phosphate anion was of great importance in enhancing the ionization efficiency of phosphopeptide in MALDI-MS.