EN ISO 20418-3:2020

SLOVENSKI STANDARD
01-september-2020
Tekstilije - Kvalitativna in kvantitativna proteomska analiza nekaterih živalskih
vlaken - 3. del: Odkrivanje peptida z uporabo LC-MS brez zmanjšanja proteina (ISO
20418-3:2020)
Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibres -
Part 3: Peptide detection using LC-MS without protein reduction (ISO 20418-3:2020)
Textilien - Qualitative und quantitative Proteomanalyse einiger Tierhaarfasern - Teil 3:
Peptiddetektion mit LC-MS ohne Proteinreduktion (ISO 20418-3:2020)
Textiles - Analyse protéomique qualitative et quantitative de certaines fibres animales -
Partie 3: Détection des peptides par LC-MS sans réduction protéique (ISO 20418-
3:2020)
Ta slovenski standard je istoveten z: EN ISO 20418-3:2020
ICS:
59.060.01 Tekstilna vlakna na splošno Textile fibres in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 20418-3
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2020
EUROPÄISCHE NORM
ICS 59.060.01
English Version
Textiles - Qualitative and quantitative proteomic analysis
of some animal hair fibres - Part 3: Peptide detection using
LC-MS without protein reduction (ISO 20418-3:2020)
Textiles - Analyse protéomique qualitative et Textilien - Qualitative und quantitative
quantitative de certaines fibres animales - Partie 3: Proteomanalyse einiger Tierhaarfasern - Teil 3:
Détection des peptides par LC-MS sans réduction Peptiddetektion mit LC-MS ohne Proteinreduktion (ISO
protéique (ISO 20418-3:2020) 20418-3:2020)
This European Standard was approved by CEN on 7 June 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20418-3:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 20418-3:2020) has been prepared by Technical Committee ISO/TC 38
"Textiles" in collaboration with Technical Committee CEN/TC 248 “Textiles and textile products” the
secretariat of which is held by BSI.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2021, and conflicting national standards shall
be withdrawn at the latest by January 2021.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 20418-3:2020 has been approved by CEN as EN ISO 20418-3:2020 without any
modification.
INTERNATIONAL ISO
STANDARD 20418-3
First edition
2020-06
Textiles — Qualitative and
quantitative proteomic analysis of
some animal hair fibres —
Part 3:
Peptide detection using LC-MS without
protein reduction
Textiles — Analyse protéomique qualitative et quantitative de
certaines fibres animales —
Partie 3: Détection des peptides par LC-MS sans réduction protéique
Reference number
ISO 20418-3:2020(E)
©
ISO 2020
ISO 20418-3:2020(E)
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2020 – All rights reserved

ISO 20418-3:2020(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 2
5 Principle . 2
6 Reagents . 3
7 Apparatus . 3
8 Test method . 4
8.1 Sampling . 4
8.2 Preliminary identification . 4
8.3 Wash for degreasing . 4
8.4 Powderization of fibres . 4
8.5 Trypsin digestion . 4
8.6 Marker peptides . 5
8.7 LC-MS analysis . 5
8.8 Evaluation of the validity of observed data . 5
8.9 Calculation of correction factor . 5
8.9.1 Correction factor . 5
8.9.2 Correction factor for Cas1 against She1 and Yak1 (kc). 6
8.9.3 Correction factor for Yak2 against She2, She3 and Cas2 (ky) . 6
8.9.4 Correction factor for alpaca and camel (ka) . 6
8.9.5 Correction factor for angora (kr) . 6
8.10 Calculation of blending ratio . 6
8.10.1 Calculation of blending ratio of cashmere, sheep wool and yak . 6
8.10.2 Calculation by She1, Cas1, Yak1 and kc . 7
8.10.3 Calculation by She2, She3, Cas2, Yak2 and ky . 7
8.10.4 Calculation by She1, She2, She3, Cas2, Yak1 . 7
8.10.5 Calculation of blending ratio of camel, alpaca and angora . 8
9 Test report . 9
Annex A (informative) Marker peptides of cashmere, sheep wool and yak fibres .10
Annex B (informative) Example of LC-MS analysis conditions .13
Annex C (informative) Analysis of camel, alpaca and angora fibres .16
Annex D (informative) Example of marker peptide mass chromatograms .19
Annex E (informative) Results of interlaboratory study .23
Bibliography .24
ISO 20418-3:2020(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 38, Textiles, in collaboration with the
European Committee for Standardization (CEN) Technical Committee CEN/TC 248, Textiles and textile
products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).
A list of all parts in the ISO 20418 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved

ISO 20418-3:2020(E)
Introduction
Cashmere is a long slender fibre obtained from cashmere goats and is expensive because of its high
quality and rarity. Mislabelling or adulteration of cashmere products blended with other cheaper
animal fibres such as sheep wool and yak have been repeatedly reported worldwide.
Current official methods to identify specific animal fibres are based on microscopic observations.
However, the microscopy-based identification is becoming increasingly difficult due to a wider use
of chemical or physical treatments in the manufacturing process. Given these issues, several other
methods have also been studied either to distinguish fibre structures by the use of near-infrared
spectroscopy or terahertz spectroscopy, or to distinguish DNA sequences by the use of polymerase
chain reaction. Nevertheless, each method has shown some complications when applied. Therefore, it is
required to develop novel identification methods.
Animal fibres consist mainly of proteins called keratins and some associated proteins. Therefore, the
most promising methods to identify fibres are based on the analysis of proteins contained in textiles.
Commonly, proteins are analysed by being subjected to digestion by trypsin, resulting in smaller
molecules, i.e. peptides, which will be later characterized through mass spectrometry. Accordingly,
identification methods using either matrix-assisted laser desorption/ionization time-of-flight mass
spectrometer or liquid chromatography/mass spectrometer (LC-MS) have been studied. When
comparing these options, the latter type of instrument is less expensive and more readily available in
testing laboratories as a versatile analytical instrument than the former. Moreover, LC-MS has a high
quantitative capability, and is therefore preferable to calculate the blending ratio of animal fibres.
Keratins are highly insoluble due to the disulphide bonds they tend to form, both at an intramolecular
as well as at an intermolecular level. Thus, keratins are generally extracted in the presence of reducing
agents. However, this reducing step is considered as time-consuming and arduous. In this document,
an alternative method in which cysteine-free peptides are selected for identification markers is used,
thereby eliminating the need of the reducing step and enabling rapid preparation of LC-MS samples.
Both ISO 20418-1 and this document describe procedures using LC-MS, but they differ regarding the
method utilized to extract the peptides. In ISO 20418-1, proteins are first extracted from fibres with
a thiourea/urea/dithiothreitol (DTT) solution, and then digested by trypsin to obtain peptides. In the
process described here, peptides are directly extracted by trypsin digestion of mechanically powdered
fibres. The method has been shown to be useful even for highly processed samples and is applicable to
various types of animal hairs such as goat (cashmere or mohair), wool and yak.
INTERNATIONAL STANDARD ISO 20418-3:2020(E)
Textiles — Qualitative and quantitative proteomic analysis
of some animal hair fibres —
Part 3:
Peptide detection using LC-MS without protein reduction
1 Scope
This document specifies a qualitative and quantitative procedure to determine the composition of
animal hair fibre blends (made of wool, cashmere, yak, alpaca, camel or angora) by LC-MS without
protein reduction.
NOTE 1 The composition of non-animal hair fibres can be measured by ISO 1833 (all parts). Both results are
combined to determine the total fibre composition.
The method is based on a preliminary identification, by light microscopy, of all fibres in the blend on the
[4]
basis of their morphology, according to ISO/TR 11827 . It is not applicable if fibres of the same animal
species (such as blends of cashmere and mohair) are present.
NOTE 2 In this case, the quantitative analysis is performed using microscopical analysis [for example,
ISO 17751 (all parts)].
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 1833-1, Textiles — Quantitative chemical analysis — Part 1: General principles of testing
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 17751 (all parts), Textiles — Quantitative analysis of cashmere, wool, other specialty animal fibers and
their blends
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
animal hair fibre
type of keratin fibre for textile use, such as wool, cashmere, yak, alpaca, camel or angora
3.2
Bovidae
biological family of cloven-hoofed, ruminant mammals including cashmere goat, sheep and yak
ISO 20418-3:2020(E)
3.3
Camelidae
biological family of even-toed ungulate mammals including camel and alpaca
3.4
protein
polymer of amino acids that play many critical roles in the body
3.5
peptide
small protein (3.4) consisting of approximately less than 50 amino acids
3.6
marker peptide
portion of a protein (3.4) used for its identification, recovery and purification
3.7
mass chromatogram
chromatogram for a specific mass-to-charge ratio
3.8
total ion chromatogram
TIC
chromatogram with each data point created by summing up intensities of all mass spectral peaks
belonging to the same scan
3.9
selected ion monitoring
SIM
mass spectrometry scanning mode in which only a limited m/z range is transmitted/detected by the
instrument
4 Symbols and abbreviated terms
A peak area
W blending ratio
Br Bovidae rate
Cr Camelidae rate
ka correction factor for alpaca and camel
kc correction factor for cashmere
kr correction factor for angora
ky correction factor for yak
m/z mass to charge ratio, where m is the mass, expressed in atomic mass unit, and z is the charge
number of ions
5 Principle
The mechanically powdered fibres are directly subjected to trypsin digestion without prior reduction.
The analysis of the digested peptides is performed with LC-MS. The percent composition is calculated
from the peak areas of the species-specific marker peptides.
2 © ISO 2020 – All rights reserved

ISO 20418-3:2020(E)
6 Reagents
The following analytical grade reagents shall be used.
6.1 Acetone, with purity greater than or equal to 99,5 %.
6.2 Water, grade 3 quality specified in ISO 3696.
6.3 Ammonium hydrogen carbonate (NH HCO ) solution (25 mmol/l)
4 3
— 197,5 mg of NH HCO , with purity greater than or equal to 96,0 %.
4 3
— Make up 100 ml by adding water (6.2).
6.4 Trypsin, sequencing-grade porcine trypsin modified by reductive methylation.
6.5 Acetonitrile, with purity greater than or equal to 99,8 %.
6.6 Formic acid, with purity greater than or equal to 98 %.
6.7 Trypsin solution
— Trypsin (6.4) 20 μg.
— 0,1 % formic acid (6.6) 200 μl.
7 Apparatus
The usual laboratory apparatus and, in particular, the following.
7.1 Heating mantle, capable of operating at a temperature range of 50 °C to 150 °C.
7.2 Mill, beads mill, cryogenic grinder or an equivalent, capable of crushing materials into an extremely
fine powder.
7.3 Membrane filter, for aqueous solutions, with a pore size of 0,45 µm.
7.4 Heat block, capable of heating microtubes at 37 °C.
7.5 Tube mixer, capable of vortex microtubes and LC vials for about 30 min.
7.6 Centrifugal evaporator, capable to deliver 5 000 g.
7.7 LC-MS, liquid chromatography–mass spectrometer, capable of detecting m/z (u) range from
200 m/z (u) to 1 500 m/z (u).
NOTE (u) is for unified atomic mass unit, SI unit.
7.8 LC vial, shall be glass or polymethylpentane.
7.9 LC column, octadecyl (C-18)-silica reversed phase column.
7.10 Balance, with a resolution of at least 0,001 g.
ISO 20418-3:2020(E)
7.11 Recovery flask (eggplant flask or round-bottom flask).
8 Test method
8.1 Sampling
The general requirement is that the test specimen shall be representative for the lot of material from
which it is taken. The method to obtain a fibre test specimen differs depending on the sample form. The
terms relating to sampling for the various types of samples shall be in accordance with ISO 1833-1.
8.2 Preliminary identification
The preliminary qualitative analysis of the animal ha
...