Separating Protein from Human Serum for Calcium Isotopic Analysis: Insights into Using 44 Ca/ 42 Ca as a Biomarker for Bone Metabolism Quinn Shollenberger, ASU/NASA Space Grant Mentors: Ariel Anbar, Melanie Channon, and Gwyneth Gordon University of Arizona April 12, 2014
Separating Protein from Human Serum for Calcium Isotopic Analysis: Insights into Using 44 Ca/ 42 Ca as a Biomarker for Bone Metabolism. Quinn Shollenberger, ASU/NASA Space Grant Mentors: Ariel Anbar, Melanie Channon, and Gwyneth Gordon University of Arizona April 12, 2014. Motivation. - PowerPoint PPT Presentation
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Separating Protein from Human Serum for Calcium Isotopic Analysis: Insights into Using 44Ca/42Ca as a Biomarker for Bone
Metabolism
Quinn Shollenberger, ASU/NASA Space GrantMentors: Ariel Anbar, Melanie Channon, and Gwyneth
GordonUniversity of Arizona
April 12, 2014
MotivationCa isotopes in urine were shown to track bone loss during bed rest studies
Bone’s preference for lighter isotopes is a phenomenon known as fractionation
Potential biomarker applications for detecting bone loss: multiple myeloma and other cancers that metastasize to bone, osteoporosis, long term space flight
Are there other fractionations in addition to bone that arehappening elsewhere inthe body
- Ca binding proteins in human blood serum
44Ca/42Cabone<44Ca/42Casoft tissue
Melanie Channon
make sure to mention that they are Ca isotopes in urine
Melanie Channon
for
Melanie Channon
so, it's not a proven biomarker. Rather, it shows great potential to be a useful biomarker. Therefore, if we can validate it, then it could be applied to diseases. We have results from multiple myeloma patients that shows promise, but it still hasn't been rigorously proven as of yet. Which, is what we are trying to do.
Melanie Channon
So, multiple myeloma is a blood cancer found in bone marrow that is known to causes severe lesions in bone and bone density loss. So, that is the primary cancer that we are trying to validate it for. And, then metastases of breast and prostate cancer are also useful. But, you should definitely mention multiple myeloma
Separation of Protein from the Flow Through
NanoDrop 2000 spectrophotometer used to
measure absorbance of proteins in samples. Image
from Thermo Scientific
Microseps used for protein separation. Image
from Pall Corporation
Protein Flow Through
Melanie Channon
when you mention that the nanodrop detects the protein abundance, don't forget to say why we care about that - to check for complete separation.
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5
10
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35
Protein Concentra-tion
Centrifugation Time (minutes)
Prot
ein
Conc
entr
ation
(g/d
L)
Protein samples at effective separation
Protein Concentration of Serum Samples
Methods
Samples during Calcium columns.Example of how ion-
exchange resin works.
-Protein and Flow Through samples go through digestion and column chemistry to eliminate interferences in the measurements of the calcium isotopic composition
Melanie Channon
so, it's not just getting rid of all the metals, but more specifically getting rid of metals or any elements that could cause interferences during the measurements
so, Ca is not the only specific one we want to measure. We also want to measure the ones that are most likely to cause interferences and make sure that they are low.
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45Flow Through Samples
Protein Samples
Centrifugation Time (minutes)
Amou
nt o
f Cal
cium
(mic
rogr
ams)
Cut-off for suffi-cient mass of cal-cium needed for isotopic analysis
Amount of Calcium in Protein and Flow Through Samples
Protein samples at effective separation
Flow through samples at effective separation
Final Conclusions
Centrifugation time for effective separation is 90 minutes
Flow through samples have plenty of calciumTo do
-Repeat separation with higher human blood serum concentration
-measure all samples for calcium isotopic analysis
Melanie Channon
so, you might want to be prepared for someone to ask you why there is so much more Ca in the media than in the protein. I believe that there should be 50% in each pool.So, we might need to think about the possibility that the proteins are breaking down and passing through the filter. :(
Acknowledgements
• Advisors– Ariel Anbar– Melanie Channon– Gwyneth Gordon