Fully Automatable Two-dimensional HILIC–RP Liquid Chromatography with Online Tandem Mass Spectrometry for Shotgun Proteomics

Poster PresentationConference theme: Proteomics: Better for lifeMultidimensional liquid chromatography (MDLC) which multiples the resolution power of individual dimension with high orthogonality is a very efficient front-end separation method for analyzing the digests of complex biological samples. Among the existing two dimensional liquid chromatography (2DLC) systems, the combination of hydrophilic interaction liquid chromatography (HILIC) followed by low-pH reversed-phase (RP)LC (HILIC-RP) has very high orthogonality and is a very promising 2DLC method. Herein, a fully automatable two-dimensional (2D) liquid chromatography system was developed for shotgun proteomics analyses, which coupling the hydrophilic interaction liquid chromatography (HILIC) TSKgel Amide 80 (a non-ionic type) with the low-pH reversedphase (RP) chromatography. The performance of the 2D HILIC-RP LC platform was investigated at both pH 6.8 (neutral pH) and pH 2.7 (acidic pH) of the first dimension HILIC column by duplicate analyses of a Rat pheochromocytoma lysates.Online coupling of the neutral-pH HILIC and RP systems outperformedthe acidic HILIC–RP combination,resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172unique peptides) increases in the number of identified proteins and peptides. To further test the established 2D HILIC-RP platform, we identified 2648 non-redundant proteins from triplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately41 to 106 copies per cell, which contained up to 2164 different validated protein species with a dynamic range of concentrations up to approximately 104. Herein, this studyestablished a fully automated 2D liquid chromatography platform to enable onlinecoupling of different HILIC and RP chromatography systems, thereby expanding the choice and application of multidimensional liquid chromatography for shotgun proteomics.published_or_final_versio

Fully automatable three-dimensional capillary-/nano-flow liquid chromatography for shotgun proteomics: a reversed-phase/strong cation exchange/reversed-phase approach

Poster presentation - Proteomics: Sample Preparation and Separations: TP 146This journal supplement is the 60th ASMS Conference ProgramThe 60th ASMS Conference on Mass Spectrometry and Allied Topics, Vancouver, Canada, 20-24 May 2012. In Journal of The American Society for Mass Spectrometry, 2012, v. 23 suppl. 1, p. S92, abstract no. TP 14

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

We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10 6 copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10 4. This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex

Adrenomedullin Enhances Invasion of Human Extravillous Cytotrophoblast-Derived Cell Lines by Regulation of Urokinase Plasminogen Activator Expression and S-Nitrosylation

Supported by funding from the University of Hong Kong (code 104106)Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM22–52 (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P < 0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.link_to_OA_fulltex

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 52h. 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. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex

N-Linked Glycoprotein Analysis Using Dual-Extraction Ultrahigh-Preformance 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

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. © The Royal Society of Chemistry 2011.link_to_subscribed_fulltex

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 25μg 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. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.link_to_subscribed_fulltex

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

Summary: •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. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).link_to_OA_fulltex