ABSTRACT
A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.
Subject(s)
CDC28 Protein Kinase, S cerevisiae/metabolism , Cell Cycle Proteins , Cyclin B/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins , Adaptor Proteins, Signal Transducing , Adenosine Triphosphatases , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Base Sequence , Blotting, Western , CDC28 Protein Kinase, S cerevisiae/chemistry , CDC28 Protein Kinase, S cerevisiae/genetics , CDC28 Protein Kinase, S cerevisiae/isolation & purification , Calmodulin/metabolism , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Chromatography, Affinity/methods , Cyclin B/genetics , Cyclin B/isolation & purification , Cyclin-Dependent Kinase Inhibitor Proteins , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/isolation & purification , HSP70 Heat-Shock Proteins/metabolism , Hemagglutinins/genetics , Hemagglutinins/immunology , Histidine/genetics , Histidine/metabolism , Macromolecular Substances , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Mutagenesis, Insertional , Nickel/metabolism , Precipitin Tests , Protein Binding , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage. To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry. Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA. Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA Damage/physiology , Genes, cdc/physiology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae Proteins , Cell Cycle/genetics , Checkpoint Kinase 2 , Chromatin/genetics , DNA Replication/physiology , DNA, Fungal/physiology , Histones/metabolism , In Vitro Techniques , Molecular Chaperones , Phosphorylation , Protein Binding/genetics , YeastsABSTRACT
We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.
Subject(s)
Mass Spectrometry/methods , Ribosomal Proteins/analysis , Saccharomyces cerevisiae/chemistry , Algorithms , Amino Acid Sequence , Chromatography, Liquid , Humans , Molecular Sequence Data , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomes/chemistry , Saccharomyces cerevisiae/genetics , Sensitivity and SpecificityABSTRACT
A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein-protein fusion product, and specific interaction with a macromolecular complex. The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.
Subject(s)
Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Proteins/analysis , Databases, Factual , Molecular Weight , Peptide Mapping , SoftwareABSTRACT
We report time-of-flight mass spectra of test mixtures of six single-stranded DNA segments. The segments range in size from 8 to 60 nucleotides (molecular weight range 2413 to 18,602 Da). The best mass spectra were obtained by pulsed laser ablation of thin frozen films of an aqueous solution of the mixture from an oxidized copper substrate. These mass spectra are dominated by the molecular-ion peak for each DNA segment, and show little evidence of fragmentation, peak broadening or cluster formation. In contrast, mass spectra obtained using UV laser ablation from an anthranilic acid matrix yield broad peaks with evidence of fragmentation, and DNA segments longer than 26 nucleotides are difficult to detect.
