Design Before You Digest: 5 Questions to Answer Before You Send Samples

The most expensive mistake in proteomics happens before a single sample reaches the mass spectrometer.

After more than twenty years of running proteomics for other researchers, I’ve noticed a pattern. The studies that disappoint rarely fail at the instrument. They fail months earlier, when samples are collected without a clear plan, and by the time anyone notices, the samples can’t be collected again.

Here are the five questions I ask every researcher before a project starts. If you can answer them all, your study is in good shape.

1. What Exactly Is Your Question?

“What’s different between these groups?” and “How much of these five proteins is there?” sound similar but call for very different experiments. The first is a discovery study, which casts a wide net across thousands of proteins. The second is a targeted study, which measures a short list of known proteins very precisely. Knowing which one you need shapes everything that follows, from sample size to cost.

2. How Many Biological Replicates Do You Have?

Running the same sample three times is not n = 3. Those are technical replicates, and they only tell you how consistent the measurement is. Biological replicates, meaning different animals, patients or independently grown cultures, tell you whether a difference is real.

Because proteomics measures thousands of proteins at once, small studies can only detect large changes. Three biological replicates per group is a bare minimum for well-controlled lab models. Human samples vary far more, so they usually need many more. A small pilot study is often the best way to find out how many you need.

3. Are Your Groups Balanced and Randomised?

If every control sample is prepared on Monday and every patient sample on Friday, you can no longer tell the difference between disease and day of the week. This is called a batch effect, and it is one of the most common reasons proteomics results can’t be trusted.

The fix is simple if it is planned in from the start: mix the groups across preparation batches, randomise the order in which samples are run, and include a pooled quality-control sample that is measured throughout.

4. Will Every Sample Be Collected and Stored the Same Way?

Proteins change after a sample leaves the body. The type of blood tube, how long it stands before centrifuging, how many times it is frozen and thawed, and the storage temperature can all leave a bigger mark on the data than the biology you are studying.

Write a collection protocol before the first sample is taken, and make sure everyone involved follows it. Our Sample Guidelines are a good starting point.

5. Have You Budgeted for the Whole Study?

It is easy to budget for the instrument time and forget everything around it: sample preparation, data analysis, statistics and the follow-up experiments needed to confirm what you find. Keep some of each sample in reserve, too. If an interesting result appears, you will want to test it again.

Start With a Conversation

None of this is complicated, but all of it is much easier to get right before the study starts than to fix afterwards. That is why consultation and experimental design is the first of our services, and why I would rather spend an hour talking through your plan than tell you later that your samples can’t answer your question.

If you are planning a study, get in touch. Bring your answers to these five questions, or bring the questions, and we’ll work through them together.