01Follow the material from one run to the next

Mitulović and colleagues investigated peptide and protein carryover in nano LC–MS and developed cleaning measures targeting the injection path and chromatographic system. Their study demonstrates that retained material can persist beyond the run in which it was introduced.Mitulović and colleagues — Preventing carryover of peptides and proteins in nano LC–MS separations (opens in a new tab)

This differs from a contaminant already present in the newly prepared sample. The distinction matters because replacing a preparation will not necessarily remove residue in the instrument, while cleaning the instrument will not resolve contamination in every prepared vial.

The observed peptide can have the expected mass and retention behaviour because it really is that peptide. Correct identity therefore does not establish that all of its signal originated from the sample currently being reported.

02Use the receiving sample as the comparison

In an original simplified example, a high sample produces 100,000 signal units. Carryover equal to 0.1% of that signal contributes 100 units to the next run. If the next sample should produce 200 units, the observed total becomes 300.

The calculation assumes linear, additive response and is not a measured instrument performance result. Its purpose is to expose the denominator: 0.1% of the earlier signal becomes 50% of the later sample’s own signal.

A carryover criterion therefore needs a connection to the low results the method must report. Describing carryover only as a percentage of the largest injection can make its practical effect difficult to judge.

03Read blanks in their sequence context

A blank before a high sample and another after it ask different questions. If the earlier blank is clear and the later one contains the target signal, the sequence provides a reason to investigate material retained from the intervening injection.

That pattern is evidence to investigate, not proof of the precise contaminated component. A blank preparation itself can be contaminated, and a problem may involve more than one part of the system. Keep the preparation records and injection history together.

A repeat injection of the affected low sample can also be misleading if the intervening runs have altered the residue. A changed result may reflect the instrument’s recent history rather than instability of the sample.

Do not automatically subtract one blank signal from all subsequent samples. Carryover can vary with the preceding material and sequence; a correction needs evidence that it represents the contribution being corrected.

04Consider effects beyond concentration

A primary hydrogen-exchange MS study showed that carried-over peptides could create misleading two-population isotope patterns. Retained material had a different exchange history and could mimic an interpretation about protein behaviour.False EX1 signatures caused by sample carryover during HX MS analyses (opens in a new tab)

That specialised example broadens the practical concern: carryover can distort an inferred structural pattern as well as a measured amount. It does not mean every two-population spectrum is an artefact.

A useful investigation records the affected sequence, the signal in appropriate blanks, the corrective action and the evidence that later measurements are suitable. A successful wash in one published system is not a universal cleaning recipe for another.

When reading a report, ask whether carryover was evaluated at levels relevant to the result and whether any affected runs were addressed. The answer belongs to the method and run evidence, not the appearance of a tidy final peak.