1. Samantha Knott October 7th, 2017
Top-Down Proteomics: A Tool For Metastatic Breast Cancer Biomarker Discovery
Samantha Knott
October 7th, 2017
2nd Year; Chemical Biology PhD Program
Dr. Ying Ge and Dr. Keely Lab Research Assistant
Wisc-AMP Scholar and NSF GRFP Scholar

2. Hallmarks of Breast Cancer (BC)
Breast Anatomy
Most patients die of distant metastasis, not the primary tumor!
Cancer occurs when abnormal cells divide uncontrollably and destroy surrounding tissue
Metastasis occurs when it has spread to other organs; 1/3 of patients
 luminal, basal and myoepithelial. 
(Peto et al., Lancet 2012)

3. Predicting Tumor Metastasis
Few clinically accepted metastatic biomarkers
Disease free interval could be months or years
80% of patients receive chemotherapy; 40% needlessly suffer chemo side affects
Significance: Biomarkers allow for identification of patients who have developed a metastatic tumor and which may respond to a treatment. Precision medicine.
(Weigelt; Nature Reviews Cancer 2005)

4. Proteomics Suited To Identify Biomarkers Of Metastatic Transition
1 protein multiple different “decorated forms”
Proteomics is the study of looking at the entire protein cohort and all proteoforms
PTM is a chemical modification or protein “decoration”
Extremely Important in Disease P M

5. Mass spectrometry Proteomics Era
Protein

6. Top-Down Proteomics Workflow
Top Down Proteomics provides a comprehensive picture of proteins and modification changes and has never been used to look for metastatic biomarkers
Protein Extraction 1 2
Separation: Based on Protein Properties 10 20 30 40 50
Time [min]
0.0
0.5
1.0
1.5 6
x10
Intens.
How many red ones?
Protein Mixture 3
Mass Spectrometry of Whole Proteins
Mass/Charge (M/Z)
Abundance
Primary vs. Metastatic
Differences in Proteome and associated PTMs
(Candidate Biomarkers)
Data Analysis 4
Smaller Pieces for Identity

7. EXAMPLE– Cardiac Disease Biomarker
80 Da
80 Da
(Zhang et al. J. Proteome Res. 2011)

8. Specific Aims
Aim 1. Develop and utilize a top-down MS proteomics method to discover candidate biomarkers for metastatic breast cancer
Aim 2. Utilize to verify in clinical samples
Significance: 1. Precision medicine
2. Has Not Yet Been Utilized

9. Mouse Model: Metastatic BC Transition
Want to optimize method
Small amount of sample
Leads to better understanding of model
Primary BC
Metastatic BC

10. Protein Extraction: Separation, Lots of Candidates
Goals:
1) Maintain sample integrity
2) ensure accurate depiction of proteins
Part 1 Metastatic
Part 2 Metastatic
Part 1 Primary
Part 2 Primary
Marker
Accomplishments:
1) >10mg of sample
2) Separation!
3) Sample Integrity
Separate Cell
Part 1
Part 2

11. Top-Down Proteomics Workflow
Protein Extraction 1 2
Separation: Based on Protein Properties 10 20 30 40 50
Time [min]
0.0
0.5
1.0
1.5 6
x10
Intens.
How many red ones?
Protein Mixture 3
Mass Spectrometry of Whole Proteins
Mass/Charge (M/Z)
Abundance
Primary vs. Metastatic
Differences in Proteome and associated PTMs
(Candidate Biomarkers)
Data Analysis 4
Smaller Pieces for Identity

12. One Example: Histone Modifications Altered
13.41 kDA Histone H2A.Z
13.81 kDA Histone H2B Type 1
Primary
Metastatic
(A)
(B)
42 Da

13. Summary
Methods developed to identify most abundant proteins and modifications
Identified many protein variants. Example: Histone
Future Directions:
Implement targeted analysis of proteoforms
Improve separation for identification of lower abundance species
Validate methods and implement in clinical practice

14. Acknowledgements Ge Research Group Dr. Ying Ge Thank You! Members
Trisha Tucholski
Vege Cai
Bifan Chen
Yutong Jin
Zachery Gregorich
Dr. Ziqing Lin
Yang Hu
Abe Wu
Kyle Brown
Tim Tiambeng
Lichen Xiu
Stephanie Werner
Dr. Ruixiang Sun
Beini
Fang
and visiting members

15. Questions?