Beating Mass Spectrometry
Output Data into Shape:
Dr. Mia Jüllig
Data Processing, Pathway Analysis &
Visualisation
How can I help?
Metabolomics and proteomics projects generate copious amounts of
data in formats that are often difficult to manage for the individual
researcher.
This talk will show the services available to:
1) Process and convert the raw outputs to organised, ready-to-publish
tables listing proteins or metabolites with basic summary statistics
2) Pathway analysis by a range of software as well as functional
grouping
3) Visualisation of the results to maximize the impact of the key
findings for publication, will also be presented.
GC or LC data
(ASAS Mass Spec Centre)
Alternative means
of initial analysis
(e.g. Statistics
Consulting Centre)
Data Analysis • Pathway Analysis • Visualisation
Other data…
Client provided processed data…
Client provided idea
Data Analysis • Pathway Analysis • Visualisation
“Metabolomics and proteomics projects generate copious amounts of
data in formats that are often difficult to manage for the individual
researcher.”
Oh really?
Data Analysis • Pathway Analysis • Visualisation
Searched LC-MS/MS output (proteomics/iTRAQ projects):
Relative abundance of
all detected proteins
Functional grouping of proteins –
biological context
Processing of quantitative data
95% confidence intervals
(Prism)
Statistical likelihood of difference
between treatment groups (Prism)
(50,000-1,000,000 rows…)
Publication quality table
Data Analysis • Pathway Analysis • Visualisation
“Metabolomics and proteomics projects generate copious amounts of
data in formats that are often difficult to manage for the individual
researcher.”
Oh really?
There is nothing
impossible about this
but getting the hang
of it takes time…
Data Analysis • Pathway Analysis • Visualisation
Software:
Excel • Prism
J. Xu, et al., Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of cholesterolfed
rabbits, Atherosclerosis (2015), http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.001
Data Analysis • Pathway Analysis • Visualisation
Validation of proteomic findings
the old fashioned way:
Petrak et al. Proteome Science 2011, 9:69
Data Analysis • Pathway Analysis • Visualisation
Validation of proteomic findings
through MRM:
(MS Centre acquires the data,
I process and present.)
J. Xu, et al., Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of
cholesterolfed rabbits, Atherosclerosis (2015), http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.001
Data Analysis • Pathway Analysis • Visualisation
Validation of proteomic findings
through MRM:
(Martin acquires the data,
I process and present.)
J. Xu, et al., Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of
cholesterolfed rabbits, Atherosclerosis (2015), http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.001
Data Analysis • Pathway Analysis • Visualisation
Quantification and preliminary IDs (MS) of metabolomics data – MZ Mine
Final IDs (MS/MS) using NIST MS Search and suitable libraries e.g. Lipid
Blast (for lipids)
Publication quality table
The problem with tables…
Data Analysis • Pathway Analysis • Visualisation
Highlighting of relevant pathways
and functions using:
•
•
•
•
MetaboAnalyst (proteins and/or metabolites)
GO enrichment analysis (proteins)
STRING analysis (proteins)
Manual methods
MetaboAnalyst (http://www.metaboanalyst.ca/faces/ModuleView.xhtml)
MetaboAnalyst (http://www.metaboanalyst.ca/faces/ModuleView.xhtml)
metabolites
proteins
Limited to proteins
and metabolites
present in metabolic
pathways…
STRING (http://string-db.org/newstring_cgi/)
STRING (http://string-db.org/newstring_cgi/)
Limited to proteins
but includes most
proteins, not just
those in metabolic
pathways
J. Xu, et al., Modelling atherosclerosis by proteomics: Molecular changes in the ascending aortas of
cholesterolfed rabbits, Atherosclerosis (2015), http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.001
GO enrichment analysis (http://geneontology.org/)
Returns GO terms for specific gene products
Returns enriched GO terms for lists of gene products
- Biological process
- Molecular function
- Cellular component
Limited to proteins
GO enrichment analysis (http://geneontology.org/)
Go-enrichment (HP )
regulation of complement activation
regulation of humoral immune response
regulation of acute inflammatory response
blood coagulation, fibrin clot formation
Oxidative stress
regulation of response to oxidative stress
enzyme linked receptor protein signalling pathway
positive regulation of signalling
negative regulation of extrinsic apoptotic signalling pathway
regulation of protein activation cascade
positive regulation of cell communication
regulation of response to stress
Signalling
DNA & protein
regulation of response to external stimulus
response to lipid
response to other organism
response to biotic stimulus
response to external biotic stimulus
response to alcohol
response to selenium ion
DNA conformation change
DNA packaging
chromatin organization
protein-DNA complex assembly
'de novo' protein folding
Limited to proteins –
and also no real
graphic output
Data Analysis • Pathway Analysis • Visualisation
GO
Enrichment
Analysis
MetaboAnalyst
STRING
Analysis
• Publication quality tables
• Comprehensive report
• Any automated graphical output
Data Analysis • Pathway Analysis • Visualisation
Most automated outputs are
appallingly ugly…
(Your reviewer)
Data Analysis • Pathway Analysis • Visualisation
I can help turn that frown upside down.
(Your reviewer)
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Outlining workflows
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Outlining workflows
Experimental setup
Data Analysis • Pathway Analysis • Visualisation
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Outlining workflows
Experimental setup
Refinement of automated
outputs
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Outlining workflows
Experimental setup
Refinement of automated
outputs
Schematics
Data Analysis • Pathway Analysis • Visualisation
Highlighting of pathways
Outlining workflows
Experimental setup
Refinement of automated
outputs
Schematics
Illustration of novel
hypotheses
J. Xu, et al., Modelling atherosclerosis by proteomics:
Molecular changes in the ascending
aortas of cholesterolfed rabbits, Atherosclerosis (2015),
http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.001
Data Analysis • Pathway Analysis • Visualisation
Typical proteomics/metabolomics project
(Raw output  Publication quality table): 4-5 days
$2,000-$2,600
Typical proteomics/metabolomics project
(3 types of analysis): 2-3 days
$1,000-$1,500
Simple image: 0.5-1 day $260-$520
More complex images: 2-3 days…
$65 ph – to earn my keep
Data Analysis • Pathway Analysis • Visualisation
Whatever you need of the above,
I am keen as a bean…
Email is a good place to start:
m.jullig@auckland.ac.nz
You can also submit a request in iLab.