Quantitative proteomics reveal up-regulated protein expression of the SET complex associated with hepatocellular carcinoma.

We combined culture-derived isotope tags (CDITs) with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) to extensively survey abnormal protein expression associated with hepatocellular carcinoma (HCC) in clinical tissues. This approach yielded an in-depth quantitated proteome of 1360 proteins. Importantly, 267 proteins were significantly regulated with a fold-change of at least 1.

Quantitative detection of single amino acid polymorphisms by targeted proteomics.

Single-nucleotide polymorphisms (SNPs) are recognized as one kind of major genetic variants in population scale. However, polymorphisms at the proteome level in population scale remain elusive. In the present study, we named amino acid variances derived from SNPs within coding regions as single amino acid polymorphisms (SAPs) at the proteome level, and developed a pipeline of non-targeted and targeted proteomics to identify and quantify SAP peptides in human plasma.

Large scale phosphoproteome profiles comprehensive features of mouse embryonic stem cells.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells.

Comprehensive profiling of phosphopeptides based on anion exchange followed by flow-through enrichment with titanium dioxide (AFET).

For large-scale analysis of phosphorylation at proteome-wide scale, a variety of affinity-based strategies have been developed to enrich phosphopeptide. Because each method differed in their specificity of isolation, the global and unbiased enrichment of phosphopeptides remains a major technical challenge in phosphoproteomics. In the present work, we demostrate that the phosphopeptide enrichment method based on an online continuous pH gradient in a strong anion exchange column (SAX method) is highly complementary to the method based on titanium dioxide (TiO2) affinity enrichment.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira that infects humans and a wide range of animals. By combining computational prediction and high-accuracy tandem mass spectra, we revised the genome annotation of Leptospira interrogans serovar Lai, a free-living pathogenic spirochete responsible for leptospirosis, providing substantial peptide evidence for novel genes and new gene boundaries. Subsequently, we presented a high-coverage proteome analysis of protein expression and multiple posttranslational modifications (PTMs).

Concurrent quantification of proteome and phosphoproteome to reveal system-wide association of protein phosphorylation and gene expression.

Reversible phosphorylation of proteins is an important process modulating cellular activities from upstream, which mainly involves sequential phosphorylation of signaling molecules, to downstream where phosphorylation of transcription factors regulates gene expression. In this study, we combined quantitative labeling with multidimensional liquid chromatography-mass spectrometry to monitor the proteome and phosphoproteome changes in the initial period of adipocyte differentiation. The phosphorylation level of a specific protein may be regulated by a kinase or phosphatase without involvement of gene expression or as a phenomenon that accompanies the alteration of its gene expression.

Fully automatic separation and identification of phosphopeptides by continuous pH-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometry.

Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.

A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis.

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process.

Quantitative phosphoproteome profiling of Wnt3a-mediated signaling network: indicating the involvement of ribonucleoside-diphosphate reductase M2 subunit phosphorylation at residue serine 20 in canonical Wnt signal transduction.

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a.

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column.

Large-scale identification of human biliary proteins from a cholesterol stone patient using a proteomic approach.

Gallbladder bile, one of the most important body fluids, is composed of water, inorganic ions, conjugated bile salts, phospholipids, cholesterol, bilirubin, mucin and proteins. The separation and identification of bile proteins remain difficult due to the complexity of this matrix. In the present study, human gallbladder bile was obtained from a cholesterol stone patient, and the proteins were isolated and purified by dialysis, precipitation and delipidation procedures.

Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps.

A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography.

High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification.

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method.

Prefractionation of proteome by liquid isoelectric focusing prior to two-dimensional liquid chromatography mass spectrometric identification.

Due to the complexity of proteomes, developing methods of sample fractionation, separation, concentration, and detection have become urgent to the identification of large numbers of proteins, as well as the acquisition of those proteins in low abundance. In this work, liquid isoelectric focusing (LIEF) combined with 2D-LC-MS/MS was applied to the proteome of Saccharomyces cerevisiae. This yielded a total of 1795 proteins that were detected and identified by 30 fractions of protein prefractionation.

The human plasma proteome: analysis of Chinese serum using shotgun strategy.

We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20 healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1) removal of six high-abundant proteins; (2) tryptic digestion of low- and high-abundant proteins of serum; (3) separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification.

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification.

Proteomic analysis of SARS associated coronavirus using two-dimensional liquid chromatography mass spectrometry and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by mass spectroemtric analysis.

The proteomes of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and its infected Vero E6 cells were detected in the present study. The cytosol and nucleus fractions of virus-infected cells as well as the crude virions were analyzed either by one-dimensional electrophoresis followed by ESI-MS/MS identification or by shotgun strategy with two-dimensional liquid chromatography-ESI-MS/MS. For the first time, all of the four predicted structural proteins of SARS-CoV were identified, including S (Spike), M (Membrane), N (Nucleocapsid), and E (Envolope) proteins.

A high-throughput approach for subcellular proteome: identification of rat liver proteins using subcellular fractionation coupled with two-dimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis.

Four fractions from rat liver (a crude mitochondria (CM) and cytosol (C) fraction obtained with differential centrifugation, a purified mitochondrial (PM) fraction obtained with nycodenz density gradient centrifugation, and a total liver (TL) fraction) were analyzed with two-dimensional liquid chromatography tandem mass spectrometry analysis. A total of 564 rat proteins were identified and were bioinformatically annotated according to their physicochemical characteristics and functions. While most extreme alkaline ribosomal proteins were identified in the TL fraction, the C fraction mainly included neutral enzymes and the PM fraction enriched alkaline proteins and proteins with electron transfer activity or oxygen binding activity.

[A novel approach for peptide identification by tandem mass spectrometry].

High throughput scoring algorithms that are used to find the match of a tandem mass spectrum to a predicted mass spectrum of a peptide within a database have been applied in shotgun proteomics. However, these algorithms could produce a significant number of incorrect peptide identifications. Here a novel approach was developed to scoring tandem mass spectra against a peptide database, in which fragment ion probabilities, number of enzymatic termini of candidate peptides, matching quality and match pattern between experimental and theoretical spectrum were considered.

An Assembly Algorithm for DNA Sequence with Repeats.

A program for assembling DNA fragments using a new approach has been developed. In the program, a filter and a sequence alignment are used to identify the true overlap between the two fragments. Then the fragments in repeat copies are put into a repeat contig and the others are put into nonrepeat contigs.

The Feasibility of Using Proteome Expression Profile for Genome Annotation.

By investigating into the expression data from ECO2DBASE (Edition 6),the feasibility of using proteome expression profile for genome annotation was tested. Based on our newly developed CRC (cellular role cluster) method,79 proteins extracted from ECO2DBASE were clustered into 4 CRCs. Function related proteins tend to be clustered into same CRC.

The Relationship between Yeast Coexpressed Gene Clusters and Their Upstream cis-acting Elements.

DNA microarrays bring biology a new approach to study functions of genes and genomes from their expression pattern on a genomic scale. With its fully sequenced genome and newly published expression patterns available, budding yeast (Saccharomyces cerevisiae) was chosen to carry out an investigation of the relationship between gene 5' upstream cis-acting elements and expression patterns using bioinformatic tools. Results show that genes in the same cluster share common cis-acting element candidates and can be regulated by same transfactors.

Reconstruction of ABC Transporter Pathways of Archaea and Comparison of Their Genomes.

Reconstruction and comparison of metabolic pathway and regulatory network is an advanced task in genome function prediction. In this study, many bioinformatic tools were employed to reconstruct all ABC transporter pathways and predict their functional features of an archaeon, Pyrococcus abyssi, on genome scale. The comparison between ABC transporter pathways of P.