MULTIISOTOPE IMAGING MASS SPECTROMETRY MIMS

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MULTI-ISOTOPE IMAGING MASS SPECTROMETRY
(MIMS) MAPPING OF PROTEIN TURNOVER IN HAIR CELLS
REVEALS HIGHLY STABLE STEREOCILIA Dua
n-Sun Zhang1,2, Valeria Piazza1, Greg McMahon1,3,
Jason Lebeau1,3, Adam Cohen1,3, Claude
Lechene1,3 and David P. Corey1,2. 1. Harvard
Medical School, 2. HHMI, 3. National Resource for
Imaging Mass Spectrometry
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Tip links are replaced within 24 h after cutting
with BAPTA
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The entire actin core of stereocilia is renewed
every 48 hours.
cochlear HC
vestibular HC
ActinGFP
Phalloidin
rat P7 organ culture transfected by gene
gun
Rzadzinska et al., 2004
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3H-labeled amino acids leave stereocilia over
2-3 weeks
2 d
5 d
8 d
10 d
13 d
chick, injected 3H-labelled amino acids at E17,
sacrificed after 2-13 d
Pickles et al., 1996
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• How to measure and image
• global turn over rates of proteins,
• at cellular and subcellular level,
• as protein renewal takes place in the living
  animal

Heavy nitrogen (15N), generously made available
by Dr. Urey, has made possible a new technique
for the investigation of protein metabolism. R.
Schoenheimer, 1937 15N natural abundance 0.36

Multi-Isotope Imaging Mass Spectrometry
(MIMS) locates and measures molecules labeled
with a stable isotope in subcellular compartments
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Secondary Ion Mass Spectrometry (SIMS)
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MIMS allows imaging and quantification of stable
isotopes in subcellular compartments

• High collection efficiency (70-80)
• High spatial resolution (x-y lt 35 nm, z 1 nm)
• High mass resolution (M/?M 1/10000)
• Ratiometric
• measurements
• (12C15N / 12C14N)

National Resource for Imaging Mass Spectrometry
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Multi-Isotope Imaging Mass Spectrometry allows
high mass resolution
15N enrichment 12C15N/12C14N
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MIMS on Bullfrog Saccule
Adult Bullfrogs Fed 15N-leucine food
1/day Sacrificed at 1, 2, 4, 8, 16, 22, 32
days Inner ear dissected out, fixed Plastic
embedding, ultrathin sections Saccular hair
cells analyzed by MIMS
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Low Stereocilia Protein Turnover in Adult
Bullfrog Hair Cells
15N/14N ratio
Mass 26
SC
HB
OM
CF
HC
SC
5 mm
adult bullfrog fed 15N for 32 days
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Stereocilia renewal rate is less than 0.3 a day
32d-fed bullfrog

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MIMS on Mouse Cochlea
Adult mice Fed 15N-leucine food ad
libitum Sacrificed at 1, 2, 4, 8, 16, 22, 32,
86, 150 days Inner ear dissected out,
fixed Plastic embedding, ultrathin
sections Organ of Corti analyzed by MIMS
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MIMS on Mouse Cochlea
renewal
52 x 52 µm
Adult mouse, fed 5 months
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Low renewal in mouse bundle proteins
renewal
Mass 26
8d
Adult mouse
22d
32d
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Pillar cells proteins turn over slowly
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Nitrogen renewal in stereocilia is low even after
5 months
Adult Mouse
cytoplasm cuticular plate stereocilia
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Experimental differences
MIMS all the proteins adult
mice in vivo normal
Actin-GFP actin neonatal mice in
culture overexpression
8 d
Rzadzinska et al., 2004
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Acknowledgements
NATIONAL RESOURCE FOR IMAGING MASS SPECTROMETRY
BRIGHAM AND WOMENS HOSPITAL HARVARD MEDICAL
SCHOOL Claude Lechene Greg McMahon Jason
Lebeau Adam Cohen
DEPARTMENT OF NEUROBIOLOGY HARVARD MEDICAL
SCHOOL David P. Corey (HHMI) Duan-Sun Zhang
(HHMI) Louise Trakimas