Keratin contamination from the bench

Keratin contamination from the bench is shed skin, hair, wool and dust that became peptides. Those peptides fragment cleanly and, in a weak sample, they can be most of what the mass spectrometer has to talk about. The instrument is often innocent. The open tube is not. Bottom-up proteomics in plain language explains why an abundant, well-behaved protein rises to the top of an identification list. Keratin is that protein, introduced by the room.

Where the protein comes from

Human skin turns over. Gloves stop you from pressing a fingerprint into a tube and do nothing about flakes that fell before you gloved, or about a cuff that dragged across a lid. Hair sheds keratins of a different set. Wool clothing sheds fibres that carry related proteins and also act as dust magnets. Ordinary room dust is a mixture of all of this plus whatever the building ventilates in.

The proteins are robust. They survive the kind of casual contact that would never register in a plasmid prep, and they are rich in lysine and arginine, so trypsin cuts them into peptides the search engine likes. UniProt entries for the keratin family are the right place to see how many distinct chains share the name. A report that says "keratin" without a type has not finished the identification.

Powdered gloves are a double problem: the powder itself is a particle source, and the habit of powdered gloves belongs to an older bench. Powder-free gloves are the usual specification for peptide work. They are not a ritual. They are a way to stop adding solids.

Why a weak sample loses the argument

A milligram-scale lysate can contain keratin and still be dominated by the biology. An excised gel band, a bead eluate from a scarce bait, or a digest of very few cells cannot. The absolute amount of skin protein that falls into an open tube during a five-minute conversation can exceed the amount of bait you eluted. The false discovery rate will then correctly identify keratin and correctly say little else, because little else out-competed it for the picker.

That is why low-input immunoprecipitations are the samples where handling discipline pays the most. It is also why a keratin result on a strong lysate and a keratin result on a bead eluate deserve different worry. The first is a contaminant annotation. The second may be the entire dataset.

Biosafety basics for research benches covers the wider habit of treating surfaces and gloves as part of the experiment. Keratin control is the proteomics-shaped piece of that habit. It is not a biosafety level by itself.

A procedure built from closures and blanks

Work on a bench you wipe and then reserve for peptide samples. Keep a clean coat on, even if the corridor felt too warm for one. Tie hair back. Leave wool outside the immediate bench area if you can. Put tubes in a rack with lids closed. Open one tube, transfer with a filtered tip, close it, then open the next. Do not lean over an open lid to see the meniscus. Do not talk across the rack. Those rules sound like manners. They are aerodynamics. Your breath and your sleeves move air onto the liquid.

Filter tips matter because the barrel of a pipette is a history of previous liquids and previous dust. The filter is not a promise against every aerosol. It is a barrier you should actually be using while the tube is open for the shortest time you can manage.

The blank is a tube of the same buffer, opened when the samples are opened, tipped when the samples are tipped, incubated when the samples are incubated, and digested if the samples are digested. A solvent blank injected at the instrument checks the column and the needle. It does not check the bench. Both blanks have jobs. Skipping the handling blank and then blaming the mass spectrometer is how keratin becomes a myth about instruments.

protocols.io methods that bother to mention a handling blank are worth copying in that one habit. Do not copy their volumes into a different kit.

Reading the names, not the word keratin

Skin keratins of types often listed as 1, 2, 9 and 10, and hair keratins, are the usual contaminants. Cytokeratins of simple epithelia, such as types 8, 18 and 19, are expressed in many tissues and cell lines of epithelial origin. If an epithelial sample contains those cytokeratins and the handling blank does not, you may be looking at biology. If the blank contains them too, you cannot tell. If a liver study, a bacterial digest or a recombinant protein contains human skin keratins, you are looking at the room, unless a human-cell component was truly part of the design.

Search the contaminant sequences on purpose so these matches land on the right entries instead of being forced onto a sample protein with a short similar stretch. Then keep them in a contaminant section of the report. Hiding them makes the table look biological and makes the next person repeat your handling.

Blank, sample, or both

A clean instrument blank with a dirty handling blank localises the problem to the bench. A dirty instrument blank localises it to the column, the needle or a previous injection. Those are different repairs. Keratin carried over from a dirty sample is an injection and wash problem. Keratin born in the tube is a handling problem. PRIDE submissions that include a contaminant discussion are easier to trust on every other protein. The archive does not make your blank optional.

When the list is full of keratin and the biology is still in there

Sometimes bait peptides and keratin peptides share a file. The bait identification can be real. The quantitative story about scarce prey cannot, until keratin is a minor fraction of the ion current. Diluting the worry with a longer gradient does not remove the flakes already digested. Remake the sample. If a reagent blank, prepared without biological material, is full of keratin, quarantine that reagent. Buffers that lived open on a bench are the usual source.

Do not solve this by deleting keratin rows and keeping the rest without comment. Reviewers and your future self need the contaminant section. Do not solve it by switching acquisition mode and hoping mixed spectra hide the problem. The protein is still the protein.

Safety and research limits

The WHO Laboratory Biosafety Manual is the public reference for how laboratories think about procedure, waste and responsibility. Keratin control is not a biosafety level. The level for your cells or tissues remains an institutional decision. Gloves and coats used to keep skin out of a tube are not automatically the gloves and coats that satisfy a containment rule. Follow both habits when both apply.

A keratin identification is a research observation about contamination or about epithelial protein. It is not a clinical skin test and not a judgement of a person's hygiene in the moral sense. It is a judgement of a process. Write it that way.

Dust, monsoon humidity, and fibres on the bench

A building that leaks dust, and a season when humid air makes fibres cling and then drop as surfaces dry, does not change the chemistry of keratin. It shortens the time an open tube stays innocent. Wipe benches more often, close tubes faster, and do not store lids face-up. Humidity also ruins the "it looked clean" test: a film of damp dust is harder to see than a dry speck and just as digestible. In that season, a handling blank on every batch is the cheaper control. Air-conditioning filters and window habits are institutional. The lid is yours. If a power cut stops the ventilation and windows open onto a dusty road, pause open-tube work until the bench has been wiped again. That pause belongs in the method more than a new software filter does.

What to say when the sample is a small immunoprecipitation

Tell the enquiry that the sample is a low-amount immunoprecipitation, a gel band, or another low-input format, because that is when contaminant load decides whether the prey can be seen. Name the species, the bead type, whether an antibody will contribute its own proteins, and whether you already run a handling blank. Ask for a report style that keeps contaminant keratins visible and separates skin types from epithelial cytokeratins.

The protein identification by LC-MS/MS reference is the closest method page. The shotgun discovery proteomics and differential abundance references apply if the prey list will be compared across conditions. Send the input class and the blank plan with the quote request. A method can be discussed around how small the sample is. The useful answer includes handling and reporting, not only an instrument method.