Supplementary Figure 2 from Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors I and II with Picomolar Affinity

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Supplementary Figure 1 from Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors I and II with Picomolar Affinity

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Supplementary Figure 3 from Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors I and II with Picomolar Affinity

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Supplementary Figure Legends 1-3 from Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors I and II with Picomolar Affinity

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Supplementary Figure 1 from Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics

PDF file, 30KB, Sequence alignment of the IGF-II-specific antibodies.</p

Supplementary Figure Legend from Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics

PDF file, 62KB.</p

Supplementary Figure 2 from Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics

PDF file, 330KB, Schematic representation and SDS-PAGE analysis of the IGF-II-specific antibodies.</p

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

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