Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry

doi:10.1083/jcb.141.4.967

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© The Rockefeller University Press, 0021-9525/1998//967 $5.00
The Journal of Cell Biology, Volume 141, Number 4, , 1998 967-977

Articles

Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry

Philip A. Wigge^*, Ole N. Jensen, Simon Holmes^*, Sylvie Souès^*, Matthias Mann, and John V. Kilmartin^*

^* Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom; and European Molecular Biology Laboratory (EMBL), D 69117 Heidelberg, Germany

A highly enriched spindle pole preparation was prepared frombudding yeast and fractionated by SDS gel electrophoresis.Forty-five of the gel bands that appeared enriched in thisfraction were analyzed by high-mass accuracy matrix-assistedlaser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional elevengene products that had not previously been localized to thespindle pole. Immunoelectron microscopy localized eight of thesecomponents to different parts of the spindle. One of the geneproducts, 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 showdefects in chromosome segregation. This is the first reportof the identification of the components of a large cellularorganelle by MALDI peptide mapping alone.

Abbreviations used in this paper: SPB, spindle pole body; AP, alkaline phosphatase; MALDI, matrix-assisted laser desorption/ionization; HA, hemagglutinin; GFP, green fluorescent protein; ppm, parts per million; ts, temperature sensitive.

O.N. Jensen was the recipient of a European Union Biotechnology Programme postdoctoral fellowship.

P.A. Wigge and O.N. Jensen contributed equally to this work.

Address all correspondence to J.V. Kilmartin, MRC Laboratoryof Molecular Biology, Hills Rd., Cambridge, CB2 2QH, UK. Tel.:(44) 1223 402242. Fax: (44) 1223 412142. E-mail: jvk{at}mrc-lmb.cam.ac.ukand O.N. Jensen, Department of Molecular Biology, Odense University,DK-5230 Odense, Denmark. Tel.: (45) 65 57 23 68. Fax: (45)65 93 27 81. E-mail: jensen{at}pr-group.ou.dk

S. Holmes's present address is Institut Curie, 26 rue d'Ulm,75248 Paris, France.

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