Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres

A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2 M NaCl, 100 mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100 ng mL⁻¹) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.     

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.     

Zirconium oxide aerogel for effective enrichment of phosphopeptides with high binding capacity

In this study, zirconium oxide (ZrO₂) aerogel was synthesized via a green sol–gel approach, with zirconium oxychloride, instead of the commonly used alkoxide with high toxicity, as the precursor. With such material, phosphopeptides from the digests of 4 pmol of β-casein with the coexistence of 100 times (mol ratio) BSA could be selectively captured, and identified by MALDI-TOF MS. Due to the large surface area (416.0 m² g⁻¹) and the mesoporous structure (the average pore size of 10.2 nm) of ZrO₂ aerogel, a 20-fold higher loading capacity for phosphopeptide, YKVPQLEIVPN[pS]AEER (MW 1952.12), was obtained compared to that of commercial ZrO₂ microspheres (341.5 vs. 17.87 mg g⁻¹). The metal oxide aerogel was further applied in the enrichment of phosphopeptides from 100 ng nonfat milk, and 17 phosphopeptides were positively identified, with a 1.5-fold improvement in phosphopeptide detection compared with previously reported results. These results demonstrate that ZrO₂ aerogel can be a powerful enrichment material for phosphoproteome study.     

Preparation of a new type of affinity materials combining metal coordination with molecular imprinting

A new approach, combining metal-coordination with molecular imprinting technology, was developed to prepare protein-affinity materials, which showed higher specific recognition ability towards the target protein than those prepared using either metal-coordination or molecular imprinting technology.     

BPA transfer rate increase using molecular imprinted polyethersulfone hollow fiber membrane

Bisphenol A (BPA) imprinted polyethersulfone (PES) hollow fiber membrane was spun using a dry–wet spinning method, the membrane was then prepared as a filter with an effective area of 200 cm². The hollow fiber filter was employed to study the BPA transport behavior. The transport ability of the prepared hollow fiber membrane was measured using 100 μmol/l BPA aqueous solutions at a flow flux of 50 and 75 ml/min, respectively. The BPA transfer rate increased for the imprinted hollow fiber membranes due to the larger amount of binding sites, comparing with the non-imprinted one. In the present study, hollow fiber membrane and the molecular imprinting technique were combined for advanced separation and the data suggested that small molecules could transfer in the direction opposite to the concentration gradient due to different pH.     

Boronic Acid functionalized core-shell polymer nanoparticles prepared by distillation precipitation polymerization for glycopeptide enrichment

The boronic acid-functionalized core-shell polymer nanoparticles, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@4-vinylphenylboronic acid (poly(MBA-co-MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid-functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time-saving. With the poly(MBA-co-MAA)@VPBA nanoparticles, 18?glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI-TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta-aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5?nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA-co-MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis.     

Cell-imprinted polydimethylsiloxane for the selective cell adhesion

A cell-patterned substrate with aptamer functionalization was prepared, which holds promise in selective cell isolation fields such as the isolation of the circulating tumor cells.     

Piperazines for peptide carboxyl group derivatization: effect of derivatization reagents and properties of peptides on signal enhancement in matrix-assisted laser desorption/ionization mass spectrometry

Piperazine-based derivatives, including 1-(2-pyridyl)piperazine (2-PP), 1-(2-pyrimidyl)piperazine (2-PMP), 1-(4-pyridyl)piperazine (4-PP), and 1-(1-methyl-4-piperidinyl)piperazine (M-PP), were used for the derivatization of carboxyl groups on peptides with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt) as coupling reagents, and trifluoroacetic acid (TFA) as activator. Taking synthetic peptides RVYVHPI (RI-7) and APGDRIYVHPF (AF-11) as samples, the yields of derivatized peptides by 2-PP, 2-PMP and 4-PP were higher than 94%. The effect of piperazine derivatives on the signals of tryptic digests of α-transferrin and bovine serum albumin (BSA) was investigated, and it was found that peptides derivatized by 2-PP and 2-PMP exhibited obviously improved ionization efficiency. Furthermore, comparison of identified peptides before and after derivatization showed that peptides with low molecular weight (MW) and high pI value were preferably detected after derivatization. In addition, after derivatization with 2-PP and 2-PMP, protein myelin basic protein S, 20 kDa protein, and histone H were confidently identified from the tryptic digests of two fractions of rat brain protein separated by reversed-phase high-performance liquid chromatography (HPLC), indicating the potential application of 2-PP and 2-PMP for the highly sensitive determination of peptides in comprehensive proteome analysis.     

Boronic Acid Functionalized Core–Shell Polymer Nanoparticles Prepared by Distillation Precipitation Polymerization for Glycopeptide Enrichment

The boronic acid‐functionalized core–shell polymer nanoparticles, poly(N,N‐methylenebisacrylamide‐co‐methacrylic acid)@4‐vinylphenylboronic acid (poly(MBA‐co‐MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid‐functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time‐saving. With the poly(MBA‐co‐MAA)@VPBA nanoparticles, 18 glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI‐TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta‐aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5 nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA‐co‐MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis.