Improved Titanium Dioxide Enrichment of Phosphopeptides from HeLa Cells and High Confident Phosphopeptide Identification by Cross-Validation of MS/MS and MS/MS/MS Spectra

Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate. Keywords: Phosphoproteomics • titanium dioxide • phosphopeptide enrichment • HeLa cells • tandem mass spectrometry • neutral loss sca

Improved Titanium Dioxide Enrichment of Phosphopeptides from HeLa Cells and High Confident Phosphopeptide Identification by Cross-Validation of MS/MS and MS/MS/MS Spectra

Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate. Keywords: Phosphoproteomics • titanium dioxide • phosphopeptide enrichment • HeLa cells • tandem mass spectrometry • neutral loss sca

Global Genomic and Proteomic Analysis Identifies Biological Pathways Related to High-Risk Neuroblastoma

Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1−) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1− tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma

Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory,, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated β-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography−tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line

Global Genomic and Proteomic Analysis Identifies Biological Pathways Related to High-Risk Neuroblastoma

Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1−) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1− tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma

Global Genomic and Proteomic Analysis Identifies Biological Pathways Related to High-Risk Neuroblastoma

Neuroblastoma (NB) is a heterogeneous pediatric tumor. To better understand the biological pathways involved in the development of high-risk neuroblastoma, we performed parallel global protein and mRNA expression profiling on NB tumors of stage 4 MYCN-amplified (4+) and stage 1 MYCN-not-amplified (1−) using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. A total of 1461 proteins represented by 2 or more peptides were identified from the quantitative ICAT analysis, of which 433 and 130 proteins are up- or down-regulated, respectively, in 4+ tumor compared to the 1− tumor. Pathway analysis of the differentially expressed proteins showed the enrichment of glycolysis, DNA replication and cell cycle processes in the up-regulated proteins and cell adhesion, nervous system development and cell differentiation processes in the down-regulated proteins in 4+ tumor; suggesting a less mature neural and a more invasive phenotype of 4+ tumor. Myc targets and ribosomal proteins are overrepresented in the 4+ tumors as expected; functional gene sets reported to be enriched in neural and embryonic stem cells are significantly enriched in the 4+ tumor, indicating the existence of a stemness signature in MYCN-amplified stage 4 tumor. In addition, protein and mRNA expression are moderately correlated (r = 0.51, p n = 208), and one-third of down-regulated proteins have lower mRNA expression (n = 47). Further biological network analysis revealed that the differentially expressed proteins closely interact with other proteins of known networks; the important role of MYCN is confirmed and other transcription factors identified in the network may have potential roles in the biology of NB tumor. We used global genomic and proteomic analysis to identify biologically relevant proteins and pathways important to NB progression and development that may provide new insights into the biology of advanced neuroblastoma

Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory,, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated β-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography−tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line

Evaluation of the Acid-Cleavable Isotope-Coded Affinity Tag Reagents: Application to Camptothecin-Treated Cortical Neurons

The new generation of isotope-coded affinity tag (ICAT) reagents have been evaluated by labeling an equimolar amount of bovine serum albumin (BSA) with ICAT-12C9 and ICAT-13C9, combining the mixtures, digesting them with trypsin and analyzing the digestate both by μRPLC-tandem MS and by matrix-assisted laser desorption ionization (MALDI) TOF/TOF MS. The use of 13C in place of 2H resulted in both of the labeled peptides having identical elution characteristics in a reversed-phase separation. This similarity in elution allows ICAT-labeled peptides to be effectively analyzed using a μRPLC−MALDI−MS strategy as well. All of the cysteinyl-containing tryptic peptides from BSA were identified with only a 10% variation in the relative abundance measurements between the light and heavy versions of each peptide. A facile method for the removal of contaminants that arise from the cleaved biotin moiety that otherwise interfere with downstream separations and MS analysis has also been developed. The new ICAT reagents were then applied to the analysis of a cortical neuron proteome sample to identify proteins regulated by the antitumor drug, camptothecin. Keywords: isotope-coded affinity tags • quantitative proteomics • mass spectrometry • cortical neuron

Improved Titanium Dioxide Enrichment of Phosphopeptides from HeLa Cells and High Confident Phosphopeptide Identification by Cross-Validation of MS/MS and MS/MS/MS Spectra

Enrichment is essential for phosphoproteome analysis because phosphorylated proteins are usually present in cells in low abundance. Recently, titanium dioxide (TiO2) has been demonstrated to enrich phosphopeptides from simple peptide mixtures with high specificity; however, the technology has not been optimized. In the present study, significant non-specific bindings were observed when proteome samples were applied to TiO2 columns. Column wash with an NH4Glu solution after loading peptide mixtures significantly increased the efficiency of TiO2 phosphopeptide enrichment with a recovery of up to 84%. Also, for proteome samples, more than a 2-fold increase in unique phosphopeptide identifications has been achieved. The use of NH4Glu for a TiO2 column wash does not significantly reduce the phosphopeptide recovery. A total of 858 phosphopeptides corresponding to 1034 distinct phosphosites has been identified from HeLa cells using the improved TiO2 enrichment procedure in combination with data-dependent neutral loss nano-RPLC-MS2-MS3 analysis. While 41 and 35% of the phosphopeptides were identified only by MS2 and MS3, respectively, 24% was identified by both MS2 and MS3. Cross-validation of the phosphopeptide assignment by MS2 and MS3 scans resulted in the highest confidence in identification (99.5%). Many phosphosites identified in this study appear to be novel, including sites from antigen Ki-67, nucleolar phosphoprotein p130, and Treacle protein. The study also indicates that evaluation of confidence levels for phosphopeptide identification via the reversed sequence database searching strategy might underestimate the false positive rate. Keywords: Phosphoproteomics • titanium dioxide • phosphopeptide enrichment • HeLa cells • tandem mass spectrometry • neutral loss sca

Systematic Proteome Analysis Identifies Transcription Factor YY1 as a Direct Target of miR-34a

MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by two or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3′ UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53, and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities