Edward Lau

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.

OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function

*The bacterial protein YqeH is a circularly permuted GTPase with homologs encoded by plant nuclear genomes. The rice homolog OsNOA1/RIF1 is encoded by the single-copy gene Os02g01440. OsNOA1/RIF1 is expressed in different tissues and is light-inducible. The OsNOA1/RIF1-EYFP fusion protein was targeted to chloroplasts in transgenic Arabidopsis plants. In addition, the rice homolog was able to rescue most of the growth phenotypes in an Arabidopsis rif1 mutant. *Rice (Oryza sativa) OsNOA1/RIF1 RNAi mutant seedlings were chlorotic with reduced pigment contents and lower photosystem II (PSII) efficiency. However, the expressions of the chloroplast-encoded genes rbcL, atpB, psaA and psbA were not affected. By contrast, reduced abundance of the chloroplast 16S rRNA was observed in the mutant. *Quantitative iTRAQ-LC-MS/MS proteomics investigations revealed proteome changes in the rice mutant consistent with the expected functional role of OsNOA1/RIF1 in chloroplast translation. The RNAi mutant showed significantly decreased expression levels of chloroplast-encoded proteins as well as nuclear-encoded components of chloroplast enzyme complexes. Conversely, upregulation of some classes of nonchloroplastic proteins, such as glycolytic and phenylpropanoid pathway enzymes, was detected. *Our work provides independent indications that a highly conserved nuclear-encoded cGTPase of likely prokaryotic origin is essential for proper chloroplast ribosome assembly and/or translation in plants.

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.

Can we predict temperature-dependent chemical toxicity to marine organisms and set appropriate water quality guidelines for protecting marine ecosystems under different thermal scenarios?

Temperature changes due to climate change and seasonal fluctuation can have profound implications on chemical toxicity to marine organisms. Through a comprehensive meta-analysis by comparing median lethal or effect concentration data of six chemicals for various saltwater species obtained at different temperatures, we reveal that the chemical toxicity generally follows two different models: (1) it increases with increasing temperature and (2) it is the lowest at an optimal temperature and increases with increasing or decreasing temperature from the optimal temperature. Such observations are further supported by temperature-dependent hazardous concentration 10% (HC10) values derived from species sensitivity distributions which are constructed using the acute toxicity data generated at different temperatures. Considering these two models and natural variations of seawater temperature, we can scientifically assess whether applying an assessment factor (e.g. 10) to modify water quality guidelines of the chemicals can adequately protect marine ecosystems in tropics, subtropics and temperate regions, respectively.

Temperature‐dependent acute toxicity of methomyl pesticide on larvae of 3 Asian amphibian species

Relative to other animal taxa, ecotoxicological studies on amphibians are scarce, even though amphibians are experiencing global declines and pollution has been identified as an important threat. Agricultural lands provide important habitats for many amphibians, but often these lands are contaminated with pesticides. The authors determined the acute toxicity, in terms of 96-h median lethal concentrations, of the carbamate pesticide methomyl on larvae of 3 Asian amphibian species, the Asian common toad (Duttaphrynus melanostictus), the brown tree frog (Polypedates megacephalus), and the marbled pygmy frog (Microhyla pulchra), at 5 different temperatures (15 °C, 20 °C, 25 °C, 30 °C, and 35 °C) to examine the relationships between temperature and toxicity. Significant interspecific variation in methomyl sensitivity and 2 distinct patterns of temperature-dependent toxicity were found. Because high proportions of malformation among the surviving tadpoles were observed, a further test was carried out on the tree frog to determine effect concentrations using malformation as the endpoint. Concentrations as low as 1.4% of the corresponding 96-h median lethal concentrations at 25 °C were sufficient to cause malformation in 50% of the test population. As the toxicity of pesticides may be significantly amplified at higher temperatures, temperature effects should not be overlooked in ecotoxicological studies and derivation of safety limits in environmental risk assessment and management.

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.

Sample Preparation for Glycoproteins

This chapter describes the methods that have been used in glycoprotein sample preparation, with a recurring theme being the separation and enrichment of glycans by exploiting their chemical and biological properties to reduce overall sample complexity. These strategies involve extracting the total protein content of the cell, isolating the glycoconjugate of interest, enzymatic digestion, purification of glycopeptides, and/or glycans. In addition, the techniques used to improve analytical detectability and sensitivity are also mentioned. The chapter also discusses the application of mass spectrometry for the analysis of glycoproteins, including the determination of glycosylation sites and glycan structures.