Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn

We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO₂) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches

Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn

We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO₂) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches

Large-Scale Identification of Phosphorylation Sites for Profiling Protein Kinase Selectivity

Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of <i>in vitro</i> kinase reaction followed by LC–MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO₂-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC–MS/MS. As a result, 3585, 4347, and 1778 <i>in vitro</i> phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites

Rapid and Deep Profiling of Human Induced Pluripotent Stem Cell Proteome by One-shot NanoLC–MS/MS Analysis with Meter-scale Monolithic Silica Columns

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis

Mentha x rubra

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis

Rapid and Deep Profiling of Human Induced Pluripotent Stem Cell Proteome by One-shot NanoLC–MS/MS Analysis with Meter-scale Monolithic Silica Columns

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography–tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98 977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more “transcription regulation”-related proteins, while the fibroblast group contained more “transport”-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis

Phosphoproteome Analysis of Formalin-Fixed and Paraffin-Embedded Tissue Sections Mounted on Microscope Slides

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC–MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases

Phosphoproteome Analysis of Formalin-Fixed and Paraffin-Embedded Tissue Sections Mounted on Microscope Slides

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC–MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases