Lianghai Hu

Profiling of Endogenous Serum Phosphorylated Peptides by Titanium (IV) Immobilized Mesoporous Silica Particles Enrichment and MALDI-TOFMS Detection

Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activities of proteins. The phosphorylation of proteins is also associated with the pathway of cancer cells. We have previously enriched the low molecular weight proteome from human plasma based on the combination of size exclusion and adsorption mechanism by using highly ordered mesoporous silica particles. Herein, highly ordered mesoporous silica particles were modified with titanium phosphonate to selectively capture the phosphopeptides from complex peptide and protein mixtures. The limit of detection for phosphopeptides from beta-casein and standard phosphopeptide spiked in human serum was as low as 1.25 fmol based on MALDI-TOFMS detection. The modified mesoporous silica particles were further used to enrich phosphopeptides from serum of hepatocellular carcinoma patients and healthy individuals and then analyzed with MALDI-TOFMS. The combination of isobaric tagging for relative and absolute quantitation labeling with MALDI-TOFMS/MS was further applied to validate the serum phosphopeptide profiling result of MALDI-TOFMS. The profiling of the serum phosphopeptides between the cancer patients and healthy persons was distinguishingly different, which indicated the potential ability of this technique for cancer diagnosis and biomarker discovery. The approach developed here would be applicable to other biological samples and a wide variety of diseases.

Determination of phenolic compounds in river water with on-line coupling bisphenol A imprinted monolithic precolumn with high performance liquid chromatography

The bisphenol A (BPA) imprinted monolithic precolumn has been prepared by in situ polymerization using 4-vinylpyridine (4-VP) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The column with good flow-through property was obtained by changing the molar ratio of the porogens (toluene and dodecanol). The selectivity and retention properties of the monolith for the BPA and other phenolic compounds were evaluated. The results show that the hydrophobic and hydrogen-bonding interaction plays important roles in the recognition process. The determination of BPA and other phenolic compounds with on-line solid-phase extraction (SPE) by monolithic precolumn coupled with conventional particulates packed and monolithic reversed-phase columns, respectively, was performed. The method was successfully applied to the analysis of phenolic compounds in river water.

Comprehensive two-dimensional high-performance liquid chromatography system with immobilized liposome chromatography column and reversed-phase column for separation of complex traditional Chinese medicine Longdan Xiegan Decoction.

A comprehensive two-dimensional HPLC system with an immobilized liposome chromatography (ILC) column in conjunction with an RP column (in tandem) was developed for the screening and analysis of the membrane-permeable compounds in a traditional Chinese medicine prescription Longdan Xiegan Decoction (LXD). More than 50 components in LXD were resolved using the developed separation system. Eight flavonoids and two iridoids were identified interacting with the ILC column; a system that mimics biomembranes. The results show that the developed comprehensive two-dimensional chromatography system can be used for identifying membrane permeable natural products in complex matrixes such as extracts of traditional Chinese medicine prescriptions.

Selective enrichment of endogenous peptides by chemically modified porous nanoparticles for peptidome analysis

We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8nm) and high-surface area (i.e. 751m(2)/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.

Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography-tandem mass spectrometry

As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography-mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 microL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 microL human serum sample.

Recent advances in mass spectrometry-based peptidome analysis

The peptidome, which is the low-molecular-weight subset of the proteome, has attracted increasing attention in recent years. However, with the interference of high-abundance protein components in complex biological mixtures (e.g., serum), selective extraction of endogenous peptides is the first and most important step before analyzing the peptidome. A number of methods and technologies have now been developed for the selective enrichment, fractionation, quantitative analysis of the endogenous peptides and their application in the potential biomarker discovery. This review will cover the methods and technologies developed in recent years for the peptidome analysis on the selective extraction, multidimensional separation and quantitative analysis, as well as their application for clinical diagnosis and biomarker discovery. The future prospects of the peptidome are also discussed.

Biological fingerprinting analysis of traditional Chinese medicines with targeting ADME/Tox property for screening of bioactive compounds by chromatographic and MS methods.

Traditional Chinese medicines (TCMs) are attracting increased global attention because of their potential to provide novel therapeutic agents based on substantial historical records of efficacy in man. Many strategies have been designed for the screening and selection of bioactive compounds from these complex natural products mixtures. Biological fingerprinting analysis (BFA), based on small molecule-biomacromolecule interactions in complex systems, has been applied to screen the multiple bioactive compounds in natural products. Here we review the chromatographic and MS approaches used for BFA of natural products with targeting absorption, distribution, metabolism, elimination and toxicity (ADME/Tox) properties. Such chromatographic methods cover a wide range of applications including liposome, serum proteins, liver homogenate and DNA profiling. MS methods for the characterization of molecular interactions between natural products and target molecules by ESI and MALDI-TOF MS are also discussed.

Improvement of performance in label-free quantitative proteome analysis with monolithic electrospray ionization emitter

The postcolumn void volume, which is introduced by the connecting tubing and void ESI emitter in the nanoflow LC coupled with MS/MS system (microLC-MS/MS), is harmful for the analysis of peptides in shotgun proteome analysis. A new type of porous C12 monolithic ESI emitter was prepared to eliminate the disruption and mixing effects occurring in the connecting tubing and void emitter. It was demonstrated that the porous hydrophobic monolith inside the emitter played a key role in retaining the good peak profile, and the average peak capacity of the whole separation system increased 12.8% in contrast to commercially available void emitter. Then, the porous C12 monolithic emitter was applied in label-free quantitative proteome analysis of two standard protein mixtures that were spiked into the tryptic digest of mouse livers extract. Compared to commercially available void ESI emitter, the number of proteins with reliable results in quantification increased greatly. And the relative quantities of the four standard proteins were all determined with the relative error < or = 6.8%. However, quantitative information of only three standard proteins could be obtained when void emitter was used.

Analysis of the human urine endogenous peptides by nanoparticle extraction and mass spectrometry identification

Peptides in urine are excreted by kidney from the blood and tissues, which are composed of a large amount of hormones, cytokines, regulatory factors and the metabolized fragments of proteins. The peptide distribution in urine will reflect the physiological and pathophysiological processes in body. In past, limited information was reported about the composition of the peptides in urine. One possible reason is that the peptides in urine are fairly low abundant and there are high concentrations of salts and organic metabolites in the urine. In this report, we extracted the peptides from human urine by highly ordered mesoporous silica particles with the pore size of 2 nm, which will exclude the high molecular weight proteins over 12 kDa. The extracted peptides were then separated into fractions according to their molecular weight by size exclusion chromatography. Each of the fractions was further analyzed by MALDI-TOF MS and μRPLC-MS/MS. Totally, 193 peptides were identified by two-dimensional SEC/μRPLC-MS/MS analysis. By analyzing the progenitor protein of the peptides; we found that two-thirds of the proteins differed from the reported urine proteome database, and the high abundant proteins in urine proteome were less detected in the urine peptidome. The developed extraction and separation methods were efficient for the profiling of the endogenous peptides in human urine. The peptidome in human urine was complementary to the human urinary proteome and may provide an emerging field for biomarker discovery.

Analysis of human serum phosphopeptidome by a focused database searching strategy

As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability.