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Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
INTRODUCTION TO PROTEOMICS II
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Outline
• Overview • Workflow • Challenges • Experimental design • Tradeoffs • Statistics • Case studies
Terminology
• Differential expression • Targeted vs. Discovery • Dynamic range • Inference • Bias • Variability • Normalization • Blocking • Randomization
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
‘Omics Technologies
Copyright © by International & American Associations for Dental Research
Mass Spectrometry-Based ‘Omics
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Raltelaar, et al., Nature Reviews Genetics 14, 35-48 (January 2013) | doi:10.1038/nrg3356
Som
e ty
pes o
f pro
teom
ics s
tudi
es
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Adapted from Walther T, Mann M. JCB 2010;190:491-500
Typical Proteomics Pipeline
Sample Prep
Mass Spec
Data Analysis
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
After a Decade - Potential Unmet
• Biological – Diverse – Ever changing – Dynamic Range
• Complex Workflow – Sample Preparation – Mass Spectrometry – Data Analysis
Bantscheff et al., 2011
Proteins in Sample
Proteins Identified
Proteins Quantified
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Mass Spec + Inference
• The “Inference Problem” • If we don’t find protein, it doesn’t mean it’s
not there • Identification and quantification requires
statistics – which requires experimental design
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Experimental Design
• Experimental design is the part of statistics that you carry out before you can carry out an experiment
• Proper planning can save many headaches • You should design your experiments with a
particular biological question in mind
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Adapted from Walther T, Mann M. JCB 2010;190:491-500
Typical Proteomics Pipeline
Sample Prep
Mass Spec
Data Analysis
Expe
rimen
tal D
esig
n
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Design Choices
• Discovery vs. Targeted • Workflow Technology • Tradeoffs • Statistical Rigor
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Nature Biotechnology ISSN: 1087-0156
Disc
over
y vs
. Tar
gete
d
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
http://www.sepscience.com/Sectors/Proteomics/Articles/460-/Advances-in-Clinical-Proteomics--A-Workshop-Report
Number Analytes vs. Number Samples
(Targeted)
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Mallick & Kuster, Nature Biotechnology, 28, 695–709. (2010) doi:10.1038/nbt.1658
Wor
kflo
w Te
chno
logy
Sample Prep
Mass Spec
Data Analysis
Patient Group A Patient Group B
Prepare Samples
Peptides
Preprocess
Quantify Identify
Analyze Statistically
MS1 & MS2
Spectra
Differentially Abundant
Peptides/Proteins
HPLC-MS/MS
Population Biological
Sample Handling
Instrument
Sources of Bias and Variability
Typical Discovery Experiment
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Why Variance Matters
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Avoiding Bias & Variability in Results
• Appropriate number of biological samples • Appropriate number of technical Replicates • Normalization • Blocking • Randomization Choices depend on workflow / technology choices
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Institute of Biostatistics and Analyses Masaryk University
Normalization
Randomization
Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279
Before you start …
Consult experts & statisticians
Plan, Plan, Plan!!!!