S. O. Siu

Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: A reversed-phase-strong cation exchange-reversed-phase approach

Previously, we described an online high-/low-pH RP-RP LC system exhibiting high-throughput, automatability, and performance comparable with that of SCX-RP. Herein, we report a variant of the RP-RP platform, RP-SCX-RP, featuring an additional SCX trap column between the two LC dimensions. The SCX column in combination with the second-dimension RP can be used as an SCX-RP biphasic column for trapping peptides in the eluent from the first RP column. We evaluated the performance of the new platform through proteomic analysis of Arabidopsis thaliana chloroplast samples and mouse embryonic mouse fibroblast STO cell lysate at low-microgram levels. In general, RP-SCX-RP enhanced protein identification by allowing the detection of a larger number of hydrophilic peptides. Furthermore, the platform was useful for the quantitative analyses of crude chloroplast samples for iTRAQ applications at low-microgram levels. In addition, it allowed the online removal of sodium dodecyl sulfate and other chemicals used in excess in iTRAQ reactions, avoiding the need for time-consuming offline SCX clean-up prior to RP-RP separation. Relative to the RP-RP system, our newly developed RP-SCX-RP platform allowed the detection of a larger number of differentially expressed proteins in a crude iTRAQ-labeled chloroplast protein sample.

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.

Differential actions of glycodelin-A on Th-1 and Th-2 cells: a paracrine mechanism that could produce the Th-2 dominant environment during pregnancy

BACKGROUND The maternal-fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear. METHODS GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting. RESULTS Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA up-regulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL. CONCLUSIONS The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during pregnancy.

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples

In this paper, we describe an online combination of reversed‐phase/reversed‐phase (RP–RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP–RP portion of this system provides comprehensive 2‐D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP–RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11‐protein mixture, we found that the system could efficiently separate native peptides and released N‐glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP–RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A‐extracted glycoproteome from human serum; in total, 134 potentially N‐glycosylated serum proteins, 151 possible N‐glycosylation sites, and more than 40 possible N‐glycan structures recognized by concanavalin A were simultaneously detected.

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.

Combinatorial use of offline SCX and online RP–RP liquid chromatography for iTRAQ-based quantitative proteomics applications

Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.

N-Linked Glycoprotein Analysis Using Dual-Extraction Ultrahigh-Performance Liquid Chromatography and Electrospray Tandem Mass Spectrometry

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.