Controlling or at least measuring

Variability In a core facility environment

Variability• Increased variability = decreased power

• Power = probability of find an effect that is there

• You can fight this by increasing the sample size but

often it is much cheaper to decrease variability

instead

Common sources of variability

• Biological

• Sample preparation

• Technical

• Data Analysis

What is possible in a core facility?

• Almost no one who sends me samples has enough

money to measure variability or wants to pay for it

• What are the best ways to communicate these

issues to customers?

• How do you know what your variability is if there are

no resources to measure it?

• How do you measure variability when you have a

large number of different types experiments?

• How much QC do you bundle into your costs if you

have to charge people

Some issues I routinely have

• Analyzing samples over months at a time….

• Sample preparation of Plant tissue may be

completely different than human cells or Plasma, or

Milk In terms of how consistently you can prepare it

• How do I know how consistently I can prepare a

sample

• Often I have no control over how the sample is

prepared. How do I deal with that?

Common ways to decrease variability

during sample prep• Process all samples on the same day by the same

persono Person can still get tired or make mistakes…Variability may not be

consistent beginning to end

o May not be possible

• Use Robotics for part of the sample prepo Many things still cannot be done well by robots

• In gel digestion of proteins is not optimal

• Decrease the things you do to a sampleo Fractionation, precipitation, SPE

• Label proteins or peptides upstream and multiplex

Common sources of variability you may not be

thinking about

• Pipetting errorso Can be vary large for small volumes

• Eppendorf 2 ul = 12% Systemic 6% random error

o Hard to get tight cv’s on your spiked peptides

• Variability due to SPE material lotso The SPE material you use today may not be the same the next time you

buy it

• Variability due to software o manual integration

o Normalization

Empirical Nulls• Are empirical Null’s a good way to measure

variability?

Is peptide or protein fractionation worth it?

• Does the fractionation kill your power?

• Is it better not to fractionate ?

• What is the least variable fractionation method for

proteomics?

• How do you measure the variability your

fractionation causes?

Example method to measure variability

• From Chris Becker (Proteometrics)

Pooled human serum

1

2

3

4

5

n

Sample aliquots are processed

Processed samples are pooled beforeanalysis and replicates are run

Processed samples are run individually

Sample Processing