Analysis of the Saccharomyces Spindle Pole by MALDI Mass Spectrometry

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Analysis of the Saccharomyces Spindle Pole by
MALDI Mass Spectrometry

• Wigge et al., 1998, J. of Cell Biology

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Spindle Pole Body -anchored in nuclear
membrane -attachment point for microtubules
during chromosome separation -functionally
equivalent to the centrosome in higher eukaryotes
http//mcb.berkeley.edu/labs/drubin_barnes/mitspin
funct.htm
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Centrosome and spindle pole body positioning
during cytokinesis. Picture from Stephen Doxsey,
2001 Nature
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Isolation of yeast spindle pole components
Why do they treat their samples with alkaline
phosphatase? How do they pick likely spindle pole
bands?
80 bands total, of which 45 are enriched in the
SP fraction
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Spotting on a MALDI Plate
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Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy cinnamic acid
MALDI
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Principal for MALDI-TOF Mass Spectrometry
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Identification of proteins using MALDI-TOF Mass
Spectrometry

• 1) Digest each band with trypsin
• Measure masses of peptide fragments and compare
  to database
• A positive ID must have multiple peptide
  matches, be a yeast protein, and have a similar
  molecular weight to the original band

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MALDI-TOF Results
-successfully analyzed 33 bands -found 5/8 known
spindle components -found 7/9 known spindle pole
body components -11 new components identified,
verified with localization studies
What are some advantages and disadvantages of
their method?
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Protein localization to the spindle pole using
immunofluorescence
anti-HA
anti-tubulin
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ImmunoEM confirms that these proteins localize to
the spindle poles
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What is the significance of coiled coil proteins
here? Why do they choose to study Ndc80p in more
detail?
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New and improved spindle pole body diagram!