Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.
A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049-6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.
Wigge
P A
PA
Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.
Jensen
O N
ON
Holmes
S
S
Souès
S
S
Mann
M
M
Kilmartin
J V
JV
D000595:Amino Acid Sequence; D015825:Chromosomes, Fungal / Q000502:physiology / Q000648:ultrastructure; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D016208:Databases, Factual; D006801:Humans; D018386:Kinetochores; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry; D009687:Nuclear Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D019151:Peptide Library; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000502:physiology* / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D008941:Spindle Apparatus / Q000502:physiology / Q000648:ultrastructure*; D013696:Temperature
D003598:Cytoskeletal Proteins; C112472:NDC80 protein, S cerevisiae; C120529:NDC80 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins