Journal: Journal of Clinical Bioinformatics

Article Title: Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

doi: 10.1186/2043-9113-1-16

Figure Lengend Snippet: The work flow for proteomic and bioinformatics PTM analysis is illustrated . [A] Proteins isolated from kinase assays are in-solution digested into peptides using the proteases Lysyl Endopeptidase and Trypsin. The peptides containing specific post-translational modifications (phosphorylation) are enriched using different resins. Non-modified peptides are used to identify proteins. [B] Purified peptides are separated on a miniaturized reverse phase chromatography column with an organic solvent gradient. Peptides eluting from the column are ionized by electrospray at the tip of the column, directly in front of the mass spectrometer. [C] The electrosprayed ions are transferred into the vacuum of the mass spectrometer. In the mass spectrometer (MS mode) all ions are moved to the mass analyzer (ion Trap), where they are measured at high resolution. The mass analyser then selects a particular peptide ion and fragments it in a collision cell. For modified peptides, the peptide mass will be shifted by the mass of the modification, as will all fragments containing the modification, allowing the unambiguous placement of the PTM on the sequence. [D] The mass and lists of fragment masses for each peptide are scanned against protein sequence databases, resulting in a list of identified peptides and proteins. The lists of proteins and their peptides are the basis for bioinformatics analysis, in order to acknowledge improvements.

Article Snippet: In this study, MSA (multistage activation) compared to DDNLMS3 (neutral loss MS3) gave more information for the suite of phosphopeptides studied when using SIMAC coupled to the ion Trap mass spectrometer.

Techniques: Isolation, Phospho-proteomics, Modification, Purification, Reversed-phase Chromatography, Solvent, Mass Spectrometry, Sequencing

Journal: Journal of Clinical Bioinformatics

Article Title: Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

doi: 10.1186/2043-9113-1-16

Figure Lengend Snippet: The 3 phosphorylated proteins (HuR, Chain B and p38p) and the 6 phosphopeptides identified and validated (amino acid sequences below the identified proteins) when using SIMAC coupled to MAS by the LTQ ion Trap mass spectrometer are shown in this table.

Article Snippet: In this study, MSA (multistage activation) compared to DDNLMS3 (neutral loss MS3) gave more information for the suite of phosphopeptides studied when using SIMAC coupled to the ion Trap mass spectrometer.

Techniques: Mass Spectrometry, RNA Binding Assay, Ubiquitin Proteomics, Activation Assay

Journal: Journal of Clinical Bioinformatics

Article Title: Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and Bioinformatic tools

doi: 10.1186/2043-9113-1-16

Figure Lengend Snippet: The efficiency and reproducibility of the phosphopeptide purification and identification when using ~3 μg of protein kinases per each resin and/or phosphoenrichment method (SIMAC, TiO 2 and IMAC) coupled to R3/C18 and MSA-LTQ ion Trap mass spectrometer is illustrated . [A] Four triplicate experiments were carried out in order to identify the phosphopeptides. The phospho-site identifications were carried out from pooled and non-pooled assays (inter- and intra-assays) confirming a high reproducibility. The 6 phosphorylated peptides identified were isolated and validated in the four triplicate analyses, not only by Mascot (at least 4 continuously -y and -b ions matched)but also by manual inspection of all the spectra. SIMAC allowed the purification of 3 phosphorylated proteins: HuR RNA binding, p38 MAP Kinase and Trapped Ubiquitin-Like Protein Activation Complex, and 6 phosphorylated peptides related to those previously mentioned proteins. TiO 2 and IMAC allowed the isolation of 2 phoshorylated proteins: HuR RNA binding and p38 MAP Kinase, and 1 phosphopeptide related to the protein kinase HuR RNA binding. [B] SIMAC coupled to MSA allowed the identification of one more phosphopeptide compared to SIMAC coupled to DDNLMS3. Nevertheless, both strategies (SIMAC coupled to MSA and SIMAC coupled to DDNLMS3) allowed the identification of the same number of phosphorylated proteins (3). [C] and [D] Three phosphorylated proteins and six phosphopeptides were identified when using SIMAC coupled to MSA. From those three phosphoproteins identified, six phosphopeptides were identified: (a) TiO 2 coupled to MSA allowed the identification of two equal/same phosphorylated proteins and four equal/same phosphopeptides as SIMAC and (b) IMAC allowed the identification of one equal/same protein and two equal/same phosphopeptides. Thus, SIMAC is more efficient than the other tested resins for this study, while TiO 2 and IMAC corroborate the reproducibility of the phosphorylated proteins and phosphopeptides identified.

Article Snippet: In this study, MSA (multistage activation) compared to DDNLMS3 (neutral loss MS3) gave more information for the suite of phosphopeptides studied when using SIMAC coupled to the ion Trap mass spectrometer.

Techniques: Phospho-proteomics, Purification, Mass Spectrometry, Isolation, RNA Binding Assay, Ubiquitin Proteomics, Activation Assay

Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy