Controlling and Measuring Variation

in Sample Preparation and Data

Analysis in a Core Facility

Environment

Christopher Colangelo

Director of Protein Profiling

MS & Proteomics Resource

Yale University

June 17, 2014

Analytical Lab Managers Interest Group

ASMS – Baltimore, MD

Sources of Technical Variability in Quantitative LC-MS

Proteomics: Human Brain Tissue Sample AnalysisPiehowski et. al. J. Proteome Res. 2013; 12(5): 2128–2137

Step 1:

• Educate your users

– Provide them with Standard Operating Procedures

– Sample Handling Procedures

– Standard Digestion Protocols

• Educate yourself and your staff

– Training classes, Workshops, Literature, Books

• learn statistics (CV, ANOVA, Blocking, T-test)

– Journal Clubs (discuss good vs. bad research)

Define the Goal of the experiment

- For each project

- Clearly articulate a goal

- Develop methods to measure results

- Outline Sample design

- Get Statistical help at beginning!!!

- Discuss internal and external standards

- Measure and repeat

Simplify

A

B

C

D

Design, Measure, and Repeat

• Design Assays which can Measure the variability in

each step of your process

• Measure this variability in a controlled experiment

• Replicates are essential (technical, biological)

Example

Measuring Between Animal, Cage, Prep Date, and Condition

cage rat ID tissue frozen or unfrozen prep-date fractions

A

1half cortex unfrozen Day 1 Subfraction 1 and Subfraction 2

half cortex frozen Day 1 Subfraction 1 and Subfraction 2

2half cortex unfrozen Day 1 Subfraction 1 and Subfraction 2

half cortex frozen Day 1 Subfraction 1 and Subfraction 2

B

3half cortex unfrozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 2 Subfraction 1 and Subfraction 2

4half cortex unfrozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 2 Subfraction 1 and Subfraction 2

C

5half cortex frozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 3 Subfraction 1 and Subfraction 2

6half cortex frozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 3 Subfraction 1 and Subfraction 2

D

7half cortex frozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 3 Subfraction 1 and Subfraction 2

8half cortex frozen Day 2 Subfraction 1 and Subfraction 2

half cortex frozen Day 3 Subfraction 1 and Subfraction 2

PCA plot for Subfraction 1 and Subfraction 2

SWATH data from 1700 proteins

Red – Day 1, Green – Day 2, Blue – Day 3

circles: Cage A, squares: Cage B, triangles: Cage C

Solid(Filled) Rat 1,3,5, Open (No fill) Rat 2,4,6

Subfraction 2Subfraction 1

SubFraction 1 Subfraction 2

red: day1

green: day2

blue: day3

Effect of preparation conditions on ALL proteins

Clustering for Subfraction 1 and Subfraction 2

SWATH data from 1700 proteins

Day1

Day2

Day 0

PCA on Subfraction 1 (between animals, cages, or prep-dates)

red: day1

green: day2

blue: day3

Comparison of Subfraction 1 fractions between prep-dates

red: day1

green: day2

blue: day3

PCA on Subfraction 2 between prep-dates

Four steps

1. Educate

1. Define Goal

2. Simplify

3. Design, Measure

and Repeat

A B C