Mascot: The trusted reference standard for protein identification by mass spectrometry for 25 years

Posted by Ville Koskinen (March 17, 2021)

How much cashmere is in this sweater?

Textiles made from luxurious animal hair like cashmere and angora can fetch a high price. Bloomberg reported that cashmere garments alone were worth $1.4 billion in global exports in 2016. Big money attracts fraudsters, and sometimes products claiming to be cashmere could be something else, like rat fur. In this context, the International Organization for Standardization (ISO) has recently standardised a new proteomics method to identify animal fibres in textiles and quantify blending ratios, ISO 20418-3:2020.

The new standard is actually part 3 of ISO 20418. Parts 1 and 2 deal with fibre identification by microscopy and quantification using a standard proteomics approach. A couple of issues limit their usefulness. Visual microscopy presents difficulties when the fibres have been subjected to chemical treatment like dyeing. Protein reduction by DTT and subsequent alkylation with iodoacetamide can affect the reproducibility of peptide MS1 intensity measurement. However, reductive alkylation seems necessary, because animal hair fibres are composed mainly of keratins with intrinsic disulfide crosslinks.

The new method is based on research by the Biological Resource Center of the National Institute of Technology and Evaluation in Japan, published as Ataku et. al, Sen’i Gakkaishi 71(3), 141-150, 2015. (Press release from the Ministry of Economy, Trade and Industry.) The key insight is focusing on cysteine-free peptides, which avoids issues with disulfide bridges. Another innovation is powdering the sample mechanically with a bead mill without attempting to unravel and separate different types of yarn. The authors consider protein identification unnecessary as long as it’s possible to uniquely identify each species at the peptide level. So, after bead milling, the sample is digested directly with trypsin with no reductive alkylation.

The original paper considered three animal species – cashmere goat (Capra hircus), sheep (Ovid aries) and yak (Bos grunniens) – including blended fabrics. A standard Mascot search of 52 textile samples produced about 1000 peptide identifications per sample. There were several candidates for cysteine-free, unique marker peptides. The authors selected one per species based on reproducibility and clear LC separation, confirmed by analysing synthesised peptides. The new sample preparation produced consistent and reproducible peptide intensities, unaffected by dyes or physical complexity.

It’s educational to load the selected peptide sequences in Unipept’s tryptic peptide analyser, which wasn’t available to the study authors at the time. The cashmere peptide SDLEAQVESLKEELLFLK and sheep wool peptide MASLLEQALATLVK (allowing for protein N-terminal methionine) are indeed unique to those two species. The yak peptide MASLLEQALATLVR maps to Bos mutus (wild yak) and Bos taurus (bovine). There’s no danger of confusion, though; cow hair is rarely spun into yarn.

To quantify blending ratios, the marker peptide peak areas were normalised to the sum intensity of shared peptides. It’s then possible to derive formulas based on normalised peak area and weighting factors to account for differences in relative protein content, contaminants, extraction efficiency and MS sensitivity. The measured blending ratios are remarkably consistent with the earlier methods. The ISO standard extends the method and includes other commercially important species, camel (Camelus ferus), alpaca (Vicugna pacos) and angora rabbit (Oryctolagus cuniculus).

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