1、BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06Textiles - Qualitative and quantitative proteomic analysis of some animal hair fibresPart 1: Peptide detection using LC-ESI-MS with protein reductionBS EN ISO 20418-1:2018National forewordThis British Standard is the UK
2、 implementation of EN ISO 20418-1:2018. It is identical to ISO 20418-1:2018. The UK participation in its preparation was entrusted to Technical Committee TCI/80, Chemical testing of textiles.A list of organizations represented on this committee can be obtained on request to its secretary.This public
3、ation does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2018 Published by BSI Standards Limited 2018ISBN 978 0 580 89702 3ICS 59.060.01Compliance with a British Standard cannot confer immunity
4、from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2018.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN ISO 20418-1:2018EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 20
5、418-1May 2018ICS 59.060.01EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGCEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2018 CEN Ref. No. EN ISO 20418-1:2018: EAll rights of exploitation in any form and by any means reserved worldw
6、ide for CEN national MembersTextiles - Qualitative and quantitative proteomic analysis of some animal hair fibres - Part 1: Peptide detection using LC-ESI-MS with protein reduction (ISO 20418-1:2018)Textiles - Analyse protomique qualitative et quantitative de certaines fibres animales - Partie 1: Dt
7、ection des peptides par LC-ESI-MS avec rduction protique (ISO 20418-1:2018)Textilien - Qualitative und quantitative Proteomanalyse einiger Tierhaarfasern - Teil 1: Peptiddetektion mittels LC-ESI-MS mit Proteinreduktion (ISO 20418-1:2018)This European Standard was approved by CEN on 16 March 2018.CEN
8、 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 appl
9、ication 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 Man
10、agement 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvi
11、a, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.English VersionEN ISO 20418-1:2018 (E)European forewordThis document (EN ISO 20418-1:2018) has been prepared by Technical Committee ISO/T
12、C 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 Novembe
13、r 2018, and conflicting national standards shall be withdrawn at the latest by November 2018.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
14、 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, Former Yugoslav Republic of Macedonia, France, Germany, Gre
15、ece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.Endorsement noticeThe text of ISO 20418-1:2018 has been approved by CEN as EN ISO 20418-1
16、:2018 without any modification.iiBS EN ISO 20418-1:2018ISO 20418-1:2018Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Abbreviated terms 25 Reagents 26 Apparatus . 37 Test method . 37.1 Sampling . 37.2 Preliminary identification . 37.3 Sample preparation 37.4
17、 Protein extraction . 47.5 Trypsin digestion of the extracted proteins . 47.6 Tryptic peptides analysis by LC-ESI-MS 47.7 Calculation and expression of results 47.8 Test report . 5Annex A (informative) Parameters . 6Annex B (informative) Markers . 8Annex C (informative) Examples of interlaboratory r
18、esults 9Bibliography .11 ISO 2018 All rights reserved iiiContents PageBS EN ISO 20418-1:2018ISO 20418-1:2018ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is no
19、rmally 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
20、 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 t
21、he 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 docu
22、ment 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.or
23、g/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 on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information
24、 about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.This document was prepared by Technical Committee ISO/TC 38, Textiles.A list of all parts in the ISO 20418 series can be found on the I
25、SO website.iv ISO 2018 All rights reservedBS EN ISO 20418-1:2018ISO 20418-1:2018IntroductionInternational producers of textiles in cashmere and other speciality fibres have to be aware of and be able to guarantee the fibre content in order to protect consumers and defend themselves from common fraud
26、s. Therefore, it is important to have a harmonized method of analysis at an international level to avoid different interpretations of the results and related conflicts between stakeholders.The innovations in the method described in this document are objective qualitative determination of the presenc
27、e of fibres derived from animal species and quantitative assessment of the relative percentages present in blends. ISO 2018 All rights reserved vBS EN ISO 20418-1:2018This page deliberately left blankTextiles - Qualitative and quantitative proteomic analysis of some animal hair fibres Part 1: Peptid
28、e detection using LC-ESI-MS with protein reduction1 ScopeThis document specifies a qualitative and quantitative testing method to determine the content of wool, cashmere, yak fibres and their blends in textiles by microscope preliminary screening, protein extraction, enzymatic digestion and specific
29、 peptides detection using a liquid chromatography-mass spectrometer equipped with electrospray ionization source (LCI-ESI-MC).This method can be applied to relevant textile products at each process stage (i.e. from raw material to garment) with a homogeneous distribution of the components. It can be
30、 applied to different types of textile materials (e.g. staples, tops, yarns and fabrics) that contain wool, cashmere or yak fibres and their blends. The method is based on a preliminary identification of all fibres in the blend on the basis of their morphology, by light microscopy. The proteins are
31、then extracted by a thiourea/urea/dithiothreitol (DTT) solution. An enzymatic digestion by trypsin of the protein extracted from the fibres is carried out. Analysis of the specific markers is performed by LC-MS and the percent composition is calculated.This method is applicable to samples containing
32、 other kinds of fibres than wool, cashmere and yak, by combining its results with the results obtained using the ISO 1833 series and/or the ISO 17751 series.This document does not apply if fibres of the same animal species are present (e.g. blends of cashmere and mohair); in this case, the quantitat
33、ive analysis can be performed using microscopic analysis (e.g. ISO 17751 series).2 Normative referencesThe 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.
34、For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 137:2015, Wool Determination of fibre diameter Projection microscope methodISO 1833 (all parts), Textiles Quantitative chemical analysisISO 17751 (all parts), Textiles Quantitative analysis o
35、f cashmere, wool, other specialty animal fibers and their blends3 Terms and definitionsFor 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: avail
36、able at https:/www.iso.org/obp IEC Electropedia: available at http:/www.electropedia.org/INTERNATIONAL STANDARD ISO 20418-1:2018 ISO 2018 All rights reserved 1BS EN ISO 20418-1:2018ISO 20418-1:20183.1animal hair fibretype of keratin fibre for textile use: wool, cashmere and yak3.2liquid chromatograp
37、hy mass spectrometryLC-ESI-MSanalytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry3.3fibre test specimenportion taken from fibre snippets randomly cut from a laboratory sample for measuremen
38、t purposes3.4trypsin digestionpart of the sample preparation for the mass spectrometric identification which consists in enzymatically cutting protein by trypsin into a limited number of shorter fragments3.5peptideshort fragment derived from a protein after enzymatic digestion3.6marker peptideportio
39、n of a protein used for its identification, recovery and purification3.7proteomic analysissystematic identification and quantification of the complete complement of proteins of a biological system using different techniques of separation and analysis such as mass spectrometry4 Abbreviated termsWS ca
40、shmereWO woolWY yak5 ReagentsDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity.5.1 Thiourea, 99 %.5.2 Urea, 99,5 % (reagent plus).5.3 DL,-dithiothreitol (DTT) 98 % (TLC), 99 % (titration).
41、5.4 Tris, (hydroxymethyl) aminomethane (Tris), 99,9 %.5.5 Ammonium bicarbonate, (NH4HCO3) eluent additive for LC-MS.5.6 Iodoacetamide, (IAM), 99 % (NMR).2 ISO 2018 All rights reservedBS EN ISO 20418-1:2018ISO 20418-1:20185.7 Trypsin Type II, from porcine pancreas (1 000 to 2 000) units/mg dry solid
42、or with an equivalent level.5.8 Water, LC-MS for LC-MS.5.9 Hydrochloric acid, 37 % (HCl) 97 %.5.10 Acetonitrile, LC-MS for LC-MS.5.11 Formic acid, (HCOOH) reagent grade 95 %.6 ApparatusThe usual laboratory apparatus and, in particular, the following.6.1 Light microscope (LM) in accordance with ISO 1
43、7751-1.6.2 Soxhlet apparatus consisting of a condenser, an extractor, a round boiling flask and a thimble (usually made of thick filter paper) which retains the sample.6.3 Microtome in accordance with ISO 137.6.4 Thermostatic bath with controllable heating and a shaking speed range of 20 r/min to 20
44、0 r/min.6.5 Centrifuge with a speed range of 200 r/min to 20 000 r/min.6.6 Vortex mixer, with speed settings ranging from 100 r/min to 3 200 r/min.6.7 Sample concentrator under nitrogen flow, with maximum gas pressure of 2 psi, equipped with steel needles.6.8 Liquid chromatography mass spectrometer,
45、 equipped with electrospray ionization source (LC-ESI-MS).7 Test method7.1 SamplingThe general requirement is that the test specimen shall be representative for the lot of material from which it is taken. The method of obtaining a fibre test specimen differs depending upon the sample form. The terms
46、 relating to sampling for the various types of samples are given in ISO 1833-1.7.2 Preliminary identificationThe preliminary qualitative analysis of the fibre composition shall be carried out on the basis of the morphology of hair fibres by light microscopy, in accordance with ISO 17751-1.7.3 Sample
47、 preparationScour and dehair the samples. Clean them either with petroleum-ether for 2 h in a Soxhlet extractor, or by immersion. Rinse them for 1 h in water at room temperature. Then rinse them for 1 h in water at 50 C. Dry the fibres in an oven at 50 C. Condition them for 24 h at a standard atmosp
48、here of 20 C and 65 % RH. ISO 2018 All rights reserved 3BS EN ISO 20418-1:2018ISO 20418-1:2018Cut fibre snippets using a microtome device (6.3) in accordance with ISO 137:2015, 6.3. It is recommended that a representative sample have a total mass of approximatively 150 mg of fibre snippets. For frag
49、mentation of fibres, it is possible to use any instrument to guarantee the homogeneous fragmentation of samples (scissors, microtome or mill).7.4 Protein extractionAdd an extraction buffer 25 mmol/l tris, 2,4 mol/l thiourea (5.1), 5 M urea (5.2), 5 % dithiothreitol (DTT) pH 8,5 (5.3) in a ratio of 9,5 ml for 150 mg of fibres.Leave the buffer in contact with the fibres for two days at 50 C under slow stirring.Filter the solution on 5 m pore size filters and centrifuge (6.5) it at 12 000 g for 20 min at room temperature.Collect the sup
