De novo identification and quantification of single amino-acid variants in human brain.

The detection of single amino-acid variants (SAVs) usually depends on single-nucleotide polymorphisms (SNPs) database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate. In human brain tissues, we can confidently identify known and novel protein variants with diverse origins. Combined with DNA/RNA sequencing, we verify SAVs derived from DNA mutations, RNA alternative splicing, and unknown post-transcriptional mechanisms. Furthermore, quantitative analysis in human brain tissues reveals several tissue-specific differential expressions of SAVs. This approach provides a novel access to high-throughput detection of protein variants, which may offer the potential for clinical biomarker discovery and mechanistic research.

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 1.5. The proteins up-regulated in HCC tissues are involved in regulatory processes, such as the granzyme A-mediated apoptosis pathway (The GzmA pathway). The SET complex, a central component in the GzmA pathway, was significantly up-regulated in HCC tissue. The elevated expressions of all of the SET complex components were validated by Western blotting. Among them, ANP32A and APEX1 were further investigated by immunohistochemistry staining using tissue microarrays (59 cases), confirming their overexpression in tumors. The up-regulation and nuclear accumulations of APEX1 was associated not only with HCC malignancy but also with HCC differentiation in 96 clinical HCC cases. Our work provided a systematic and quantitative analysis and demonstrated key changes in clinical HCC tissues. These proteomic signatures could help to unveil the underlying mechanisms of hepatocarcinogenesis and may be useful for the discovery of candidate biomarkers.

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. The absolute concentrations of three selected SAP-peptide pairs among 290 Asian individuals were measured by selected reaction monitoring (SRM) approach, and their associations with both obesity and diabetes were further analyzed. This work revealed that heterozygotes and homozygotes with various SAPs in a population could have different associations with particular traits. In addition, the SRM approach allows us for the first time to separately measure the absolute concentration of each SAP peptide in the heterozygotes, which also shows different associations with particular traits.

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. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in 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. Moreover, we found that the flow-through fraction of either SAX or SCX is very phosphopeptide-rich, which necessitates further analysis by complementary method. Here, we developed a comprehensive phosphopeptides profiling strategy based on anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX method was used as the first separation/enrichment step, which was online coupled with LC-MS/MS. The phosphopeptides in the SAX flow-through fraction were further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome was aquired. Careful comparison of four different combination strategies reveal that the AFET method showed the advantages of more identified phosphopeptides, less mass spectrometry analysis time, as well as simple and automatic process step. It is well-suited for robust and reproducible phosphoproteomics, especially in the case of small amounts of sample.

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). Approximately 64.3% of the predicted L. interrogans proteins were cataloged by detecting 2 540 proteins. Meanwhile, a profile of multiple PTMs was concurrently established, containing in total 32 phosphorylated, 46 acetylated and 155 methylated proteins. The PTM systems in the serovar Lai show unique features. Unique eukaryotic-like features of L. interrogans protein modifications were demonstrated in both phosphorylation and arginine methylation. This systematic analysis provides not only comprehensive information of high-coverage protein expression and multiple modifications in prokaryotes but also a view suggesting that the evolutionarily primitive L. interrogans shares significant similarities in protein modification systems with eukaryotes.

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. Concurrent quantification of phosphopeptides and non-phosphorylated peptides makes it possible to differentiate cellular phosphorylation changes at these two levels. Furthermore, on the system level, certain proteins were predicted as the targeted gene products regulated by identified transcription factors. Among them, several proteins showed significant expression changes along with the phosphorylation alteration of their transcription factors. This is to date the first work to concurrently quantify proteome and phosphoproteome changes during the initial period of adipocyte differentiation, providing an approach to reveal the system-wide association of protein phosphorylation and 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 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multiphosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.

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. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.

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. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of beta-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling.

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. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.

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. The resulting proteins were divided into several aliquots. One aliquot was subjected to two-dimensional gel electrophoresis (2DE). The protein spots were then in-gel digested and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). Another aliquot was directly digested and analyzed by a combination of strong cation-exchange (SCX) and reversed-phase (RP) chromatography prior to tandem mass spectrometry (2D-LC/MS/MS). Eventually, 48 and 218 unique proteins were identified from 2DE/MS and 2D-LC/MS/MS, respectively, resulting in a total of 222 unique identified proteins. Of the 218 proteins identified by 2D-LC/MS/MS, 92 were identified based on more than one unique tryptic peptide, and, of the total 222 proteins, 98 were identified based on more than one unique tryptic peptide.

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. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is approximately 0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for large-scale protein identification using existing one-dimensional HPLC systems.

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. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis without protein fractionation. LIEF-2D-LC-MS/MS also produced improved resolution of low-abundance proteins. Furthermore, we analyzed the characteristics of proteins obtained by LIEF-2D-LC-MS/MS. 1103 proteins with CAI under 0.2 were identified, allowing us to specifically obtain detailed biochemical information on these kind proteins. It was observed that LIEF-2D-LC-MS/MS is useful for large-scale proteome analysis and may be specifically applied to systems with wide dynamic ranges.

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. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.

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 total of 944 nonredundant proteins were identified under a stringent filter condition (X(corr) > or = 1.9, > or = 2.2, and > or = 3.75, < or = C(n) > or = 0.1, and R(sp) > or = 4.0) in both pooled male and female samples, in which 594 and 622 entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206 proteins were identified with at least two distinct peptides per protein and 185 proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery.

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. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

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. In addition, a novel phosphorylated site of M protein was observed. The combination of these gel-base and non-gel methods provides fast and complimentary approaches to SARS-CoV proteome and can be widely used in the analysis of other viruses.

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. Such characteristics were more apparent in proteins identified only in the TL, C, or PM fraction. The Swiss-Prot annotation and the bioinformatic prediction results proved that the C and PM fractions had enriched cytoplasmic or mitochondrial proteins, respectively. Combination usage of subcellular fractionation with two-dimensional liquid chromatography tandem mass spectrometry was proved to be a high-throughput, sensitive, and effective analytical approach for subcellular proteomics research. Using such a strategy, we have constructed the largest proteome database to date for rat liver (564 rat proteins) and its cytosol (222 rat proteins) and mitochondrial fractions (227 rat proteins). Moreover, the 352 proteins with Swiss-Prot subcellular location annotation in the 564 identified proteins were used as an actual subcellular proteome dataset to evaluate the widely used bioinformatics tools such as PSORT, TargetP, TMHMM, and GRAVY.