We acknowledge financial support from Institute of Food research BBSRC Core Strategic Grant funds, BBSRC Project BB/J004545/1.

<ol>
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T., Ghosh, D., Beaudry, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+meat+authenticity+using+bioinformatics,+targeted+peptide+biomarkers+and+high-resolution+mass+spectrometry.">Assessment of meat authenticity using bioinformatics, targeted peptide biomarkers and high-resolution mass spectrometry.</a> Food Addit. Contam. Part A-Chem. 32, 1709-1717 (2015).</li><li>Claydon, A. J., Grundy, H. H., Charlton, A. J., Romero, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+peptides+for+horse+meat+speciation+in+highly+processed+foodstuffs.">Identification of novel peptides for horse meat speciation in highly processed foodstuffs.</a> Food Addit. Contam. Part A-Chem. 32, 1718-1729 (2015).</li><li>von Bargen, C., Brockmeyer, J., Humpf, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+Authentication:+A+New+HPLC-MS/MS+Based+Method+for+the+Fast+and+Sensitive+Detection+of+Horse+and+Pork+in+Highly+Processed+Food.">Meat Authentication: A New HPLC-MS/MS Based Method for the Fast and Sensitive Detection of Horse and Pork in Highly Processed Food.</a> J. Agric. Food Chem. 62, 9428-9435 (2014).</li><li>von Bargen, C., Dojahn, J., Waidelich, D., Humpf, H. U., Brockmeyer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Sensitive+High-Performance+Liquid+Chromatography+Tandem+Mass+Spectrometry+Method+for+the+Detection+of+Horse+and+Pork+in+Halal+Beef.">New Sensitive High-Performance Liquid Chromatography Tandem Mass Spectrometry Method for the Detection of Horse and Pork in Halal Beef.</a> J. Agric. Food Chem. 61, 11986-11994 (2013).</li><li>Montowska, M., Alexander, M. R., Tucker, G. A., Barrett, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Authentication+of+processed+meat+products+by+peptidomic+analysis+using+rapid+ambient+mass+spectrometry.">Authentication of processed meat products by peptidomic analysis using rapid ambient mass spectrometry.</a> Food Chem. 187, 297-304 (2015).</li><li>Montowska, M., Alexander, M. R., Tucker, G. A., Barrett, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Peptide+markers+for+authentication+purposes+in+raw+and+cooked+meat+using+ambient+liquid+extraction+surface+analysis+mass+spectrometry.">Rapid detection of Peptide markers for authentication purposes in raw and cooked meat using ambient liquid extraction surface analysis mass spectrometry.</a> Anal. Chem. 86, 10257-10265 (2014).</li><li>Sentandreu, M. A., Fraser, P. D., Halket, J., Patel, R., Bramley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Proteomic-Based+Approach+for+Detection+of+Chicken+in+Meat+Mixes.">A Proteomic-Based Approach for Detection of Chicken in Meat Mixes.</a> J. Proteome Res. 9, 3374-3383 (2010).</li><li>Elliott, M. H., Smith, D. S., Parker, C. E., Borchers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+trends+in+quantitative+proteomics.">Current trends in quantitative proteomics.</a> J. Mass Spectrom. 44, 1637-1660 (2009).</li><li>MacLean, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26, 966-968 (2010).</li><li>Keeton, J. T., Ellerbeck, S. M., Nunez de Gonzalez, M. T., Devine, C., Dikeman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Meat Sciences. 1, 235-243 (2014).</li><li>Montowska, M., Alexander, M. R., Tucker, G. A., Barrett, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Detection+of+Peptide+Markers+for+Authentication+Purposes+in+Raw+and+Cooked+Meat+Using+Ambient+Liquid+Extraction+Surface+Analysis+Mass+Spectrometry.">Rapid Detection of Peptide Markers for Authentication Purposes in Raw and Cooked Meat Using Ambient Liquid Extraction Surface Analysis Mass Spectrometry.</a> Anal. Chem. 86, 10257-10265 (2014).</li></ol>

The authors have nothing to disclose.

好適な標的タンパク質の選択が重要です。優れた標的タンパク質は、目的の種の対応する形態は、十分な種依存性配列変異、種特異性を有し、生物内でアクセス可能な量で存在する必要があります。処理を受けた混合物（例えば、熱処理）を評価するため、その処理に比較的免疫配列を有するタンパク質であることが望ましいです。ミオグロビンは、調理された赤身肉などの赤肉、のための良い候補であるが、唯一の可能性ではありません。標的タンパク質が決定されると、プロトコルの最も重要な部分は、タンパク質のタンパク質分解です。ミオグロビンは異なるタンパク質がよく、代替タンパク質分解プロトコルを要求することができます。 記載されているようにプロトコルは、基準精製タンパク質に基づいてセグメントを含みます。これは、保持時間ウィンドウと、適切な前駆体およびフラグメントイオンを発見することを目指しています。このセグメントは非常に有用であるが、必須ではありません。 <p関心の2種からの対応するペプチドのペアがあっても実験することなく記載されていることができますが、クラス= &quot;jove_contentは&quot;&gt;、それは時々、配列の差が消化プロファイルに劇的な影響を有している場合です。例えば、ペプチドペアVLGFHG（牛肉）とELGFQG（馬）（ 図2の1未満の勾配としてマニフェスト）異常 ​​な定量結果を与えます。後者のペプチドは、混合物中の馬のレベルの過少見積もりを引き起こし、比較的抑制KE切断から生じるためです。異なるアミノ酸で始まる対応するペプチドは、したがって、最高の回避されます。多くの場合、対応する二つのペプチドのフラグメントは、同一のアミノ酸配列を有し、行儀されているが、これは常にそうではなく、方法の開発中にチェックする必要があります。種の同定は、相対定量よりも、これらの問題に非常に敏感ではありません。 プロトコルは、4つの赤肉のために実証されています秒3。あまりにも多くのマーカーペプチドが共溶出場合、遷移ピーク形状の品質を効果的に滞留時間を減少させ、最終的に相対的な定量推定値を分解、劣化するかもしれないが、追加の肉種が、含まれ得ます。すでに利用可能な改善された機器は、これを改善します。関連する問題ではない、すべての肉は、異なるミオグロビンを有することです。例えば、馬、ロバとシマウマミオグロビンは同一であり、したがって、厳密方法を言えば牛肉に馬やロバやシマウマを検出することができるだけです。いくつかのケースでは、ミオグロビンが同一ではないにもかかわらず、いくつかの重要なペプチドは、することができます。例えば、いくつかのラムミオグロビン由来のマーカーペプチドはまた、ヤギに表示されます。 このおよび他のタンパク質ベースの定量法が直面している合併症は、タンパク質またはペプチドのレベルは、混合物中の肉のレベルに自明と同一視することがある場合、タンパク質レベルは、全ての種にわたって一定であると仮定しなければならないことです。ミオグロビンと4赤メートルのためにこれは普遍的真実ではありません食べます。一般的にレベルは豚肉は4の最低レベルを示すと、依存種です。また、ミオグロビンレベルは肉カットと動物の年齢に応じて変化します。遷移のピーク面積の比が、ミオグロビンの比率に確実にマッピングがので、実際の肉の割合へのマッピングは、混合物中の肉の可能性ソースに関する仮定の推定図です。 この作業で概説したアプ​​ローチは、他の公開の貢献から、いくつかの方法で異なっています。より典型的な経路は、異なる種のマーカーが互いに8-12,14,19とは特定の関係を有していない、その場合には、様々な異種の種依存性マーカーペプチドを同定するプロテオミクスの方法を使用することです。これとは対照的に、我々は3バリアント 、種依存性シーケンスまで関心のあるすべての種に共通するタンパク質を選択しました。別に私たちの相対定量戦略の中心であることから、これはそのサンプル利点を有しています調製ストラテジーを最適化することができます。さらに、このような対応するタンパク質は、抽出、またはそのような調理または缶詰などのサンプルの商業的処理、例えば、同様に挙動することが予想されるかもしれません。種の同定は、その後、通常、典型的には、1つまたは2配列の差異を有する密接に関連するペプチドの検出によりCPCPアプローチ種の識別が進む中で、一方、異種のマーカーペプチドの検出を介して進行します。最後に、他に1種の重量％を推定するタンパク質の定量は、従来別々に既知の標準7,14,15に基づいて、各タンパク質の絶対定量を介して進行することがあります。しかし、較正方法を必要としないCPCP法を用いて。代わりに、相対的なレベルは、完全に絶対測定ステージをバイパスし、両種からの対応する二つのペプチドの信号強度を比較することによって推定されます。究極の目標は、あの、1つの種の重量％であることからTHER、相対定量、その後CPCPは両方のより直接的かつ2つの絶対定量測定値を比較するよりも簡単です。これらの機能は、食品の不正検出の分野における急速な監視ツールとしての技術が有用なもの、洗練されたプロトコルを用いて、約2時間であることが予想される短い実験時間、に変換されます。

The European horse meat scandal of 2013, in which undeclared horse meat was found in a number of supermarket beef products1, highlights the need for testing methods capable of detecting and measuring food fraud in meat. Several technologies have been explored, especially enzyme-linked immunosorbent assay (ELISA) and DNA-based methods2. An alternative route, based on mass spectrometry, targets species-specific peptides which in turn arise from species-specific proteins. Here we outline one such peptide-based approach that offers both identification and relative quantitation of the adulterant species in a meat mixture3. 
The protocol is framed in the context of red meats and the desire to determine the presence of one in another at the level of 1% by weight, the level considered by some to represent fraudulent food adulteration as opposed to contamination4. The method relies in the first instance on identifying a protein which is nominally 'the same' in all target meats. Myoglobin, the protein responsible for the red color of meat, is a good candidate since it is abundant, relatively heat tolerant and water soluble, and has been used for species determination of meat previously5,6. The myoglobins for beef (Bos Taurus), pork (Sus scrofa), horse (Equus caballus) and lamb (Ovis aries)3, for instance, are nominally the same, as required, but their sequences are not identical. Such groups of 'similar but different' proteins, like these four myoglobins, can conveniently be described as 'corresponding proteins'. The sequence differences in these four myoglobins are species-specific: for example, the full myoglobin proteins for beef and horse, P02192 and P68082 respectively, each comprise 154 amino acids with 18 sequence differences between the two. Subject to proteolysis using trypsin these proteins produce two sets of peptides, some of which are identical, and some which show one or more species-specific amino acid differences: corresponding proteins therefore give rise to corresponding peptides. 
The CPCP approach, therefore, seeks first to identify proteins from two or more species where these proteins exhibit limited species-specific sequence variants. These are corresponding proteins. Following proteolysis, corresponding proteins give rise to peptides, some of which likewise display species-specific sequence variants inherited from the parent protein. These are corresponding peptides. The CPCP approach can be used to compare levels of two corresponding proteins in a mixed species sample by monitoring the levels of corresponding peptides.
The natural technology for the detection of known peptides is multiple reaction monitoring mass spectrometry, or MRM-MS7. Species-specific peptides yield precursor ions, which along with their mass spectrometry fragment ions, are easily itemized in advance by software tools. These lists are then used to instruct the mass spectrometer to record only specific precursor plus fragment ion pairs, called transitions. A particular target peptide is therefore identified not only by its retention time in the chromatography preceding the mass spectrometer, but also by a set of transitions sharing a common precursor ion. This is a highly selective means of detecting known peptides that makes efficient use of the mass spectrometer resource.
Other authors have used mass spectrometry to test for meat adulteration via peptide markers but from disparate proteins8-14. Using the corresponding proteins, corresponding peptides (CPCP) scheme, however, means experimental conditions can be optimized, aiding identification of the species in the mixture from known species-specific transitions. In addition, corresponding proteins and peptides will generally behave similarly in the extraction, proteolysis and detection stages. Since transition peak areas are quantitative and reproducible, ratios of peak areas arising from pairs of corresponding peptides from different species provide a direct estimate of the relative quantities of two meats in a mixture. In contrast, more traditional quantitation routes exploit calibrations based on reference materials to establish absolute quantitation14,15.
Though the protocol is outlined in the context of myoglobin and meat, proteins other than myoglobin could be used for identification and relative quantitation via the CPCP strategy in meat mixtures, though potentially with modifications to the protocol. In addition the strategy is also applicable to binary mixtures of other species sharing one or more corresponding proteins.
The starting point for the protocol is purified 'reference' myoglobin, which for some species can be purchased but which for others must be prepared by conventional size-exclusion chromatography. The procedure for preparing reference myoglobin is not included in the protocol, but is described elsewhere3. Software tools16 are used to list candidate peptides and transitions arising from myoglobins of interest. Each reference myoglobin is subjected to proteolysis and the resultant peptides analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to discover which of the candidate precursor ions and transitions are most useful, and to determine the matching peptide retention times. The outcome of this stage is a revised list of target peptides with their transitions, suitable for species determination, and a list of CPCP pairs, suitable for relative quantitation. To test real meats, sample extractions are prepared then subjected to proteolysis to generate peptides both from myoglobin and other extraneous proteins. The myoglobin-based peptides are then monitored by LC-ESI-MS/MS based on their listed transitions. The species present in a mixture are identified by the transition peaks associated with marker peptides. Estimates of the relative amounts of two meats in a binary mixture are calculated using ratios of transition peak areas. A set of test mixtures of pairs of meats will allow the ratio of peak areas for a given pair of transitions to be checked and calibrated against actual mixtures.

<table><tbody><tr><td>Uniprot database</td><td>&lt;a href=&quot;http://www.uniprot.org/&quot;&gt;www.uniprot.org&lt;/a&gt;</td><td></td><td>Freely accessible database of protein sequences</td></tr><tr><td>Skyline software</td><td>&lt;a href=&quot;http://www.skyline.gs.washington.edu/&quot;&gt;www.skyline.gs.washington.edu&lt;/a&gt;</td><td></td><td>Free software to download that enables the creation of targeted methods for proteomic studies, peptide and fragment prediction&amp;nbsp;</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td><td>O9830</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td><td>10674922</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td><td>10010010</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td><td>U5378</td><td></td></tr><tr><td>Trypsin(from bovine pancreas treated with TPCK)</td><td>Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td><td>T1426</td><td></td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td><td>F0507</td><td></td></tr><tr><td>Coomassie Plus Protein Assay Reagent</td><td>Thermo Fisher Scientific www.thermofisher.com</td><td>1856210</td><td></td></tr><tr><td>Protein standard</td><td>Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td><td>P0914</td><td></td></tr><tr><td>Ultra Turrax homogeniser T25</td><td>Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td><td>13190693</td><td></td></tr><tr><td>Edmund and Buhler KS10 lab shaker</td><td></td><td></td><td></td></tr><tr><td>Heraeus Fresco 17 Centrifuge</td><td>Thermo Fisher Scientific www.thermoscientific.com</td><td>75002420</td><td></td></tr><tr><td>Vacuum centrifuge RC 1022</td><td>Jouan</td><td></td><td></td></tr><tr><td>Plate Reader</td><td></td><td></td><td></td></tr><tr><td>Strata-X 33u polymeric reversed-phase cartridges 60 mg/3 ml tubes</td><td>Phenomenex, Macclesfield, UK</td><td>8B-S100-UBJ</td><td></td></tr><tr><td>4000 QTrap triple-quadrupole mass spectrometer</td><td>AB Sciex, Warrington, UK www.sciex.com</td><td></td><td></td></tr><tr><td>1200 rapid resolution LC system</td><td>Agilent, Stockport, UK</td><td></td><td></td></tr><tr><td>XB C18 reversed-phase capillary column (100 mm x 2.1 mm, 2.6 &amp;micro;m particle size)</td><td>Phenomenex, Macclesfield, UK www.phenomenex.com</td><td></td><td></td></tr><tr><td>Analyst 1.6.2 software</td><td>AB Sciex, Warrington, UK www.sciex.com</td><td></td><td>QTrap data acquisition and analysis, including peak area integration</td></tr><tr><td>Autosampler vials</td><td></td><td></td><td></td></tr></tbody></table>

species determination and quantitation in mixtures using mrm mass spectrometry of peptides applied to meat authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

単一の動的モードMRM実験では、各プログラムの移行が指​​定された保持時間ウィンドウにわたって（検出器秒あたりのカウント、CPSとして）別々に記録されています。したがって、1つの実験において収集されたすべてのデータから、各遷移のピーク強度は、個々に抽出することができます。そして、唯一の有限の信号は、その遷移に設定保持時間枠のためです。窓の外では、信号は定義によりゼロです。例えばいずれかの遷移のための信号、馬から752→1269（質量1,501.66ダルトンは、前駆体イオンの m / z 751.84ダルトンは、状態= 2、フラグメントイオンyの13を充電するモノアイソトピックペプチド）は、典型的には、測定ノイズとないとだけ競うために持っていますおそらく他の種に由​​来する可能性がある他の遷移ピークから。出力は、したがって、共通の前駆体イオンを共有し、それらの遷移のための共通の保持時間できれいなピークは、遷移ごとに1、のセットです。 図1は、1％の混合物のための4つのトランジション752→（1269、706、248、1366）のセットの出力/重量を示しています牛肉で馬。表示された4遷移が馬に関連付けられており、純粋な牛肉、羊肉や豚肉のサンプルに存在しないされているので、これらのピークは、馬の存在を意味します。ロバスト性の基準に応じて、ノイズレベルにある特定の信号を超える二つ以上の遷移の組それぞれは、識別を確立します。この図は、したがって、牛肉で馬ワット/ wの1％の混合物中の馬の存在を確立します。 時折、単一の孤立遷移が検出されます。これは、質量分析計にシステムから期待され、プログラムしたもので、おそらく無関係なタンパク質から、前駆イオンと単一フラグメントの偶然の一致を示します。予期しない保持時間における特異ピークの性質、及びその発生は、Siであります無視することができ、偶発的移行のgnature。 各遷移のピーク下の面積を個別に計算することができます。適切なフラグメントに基づいて、牛肉の遷移ピーク面積に対する馬の割合は、例えば、767→1299へ→1269年752（馬）（牛肉）は、混合物中の実際の肉の割合に比例します。 図2は、Aを示し牛肉と馬との混合物中の馬の重量のための重量百分率対これらの2つの遷移のピーク面積百分率のプロット。パーセンテージ遷移ピーク面積は、肉の重量のための重量パーセントと一致した場合、傾きはこのプロットにおける傾きがこれらの遷移とCPCP対について、遷移ピーク面積は、相対的な量の信頼性の尺度を与える、ことを示す、1.03 1です。混合物中の2つの肉の。他の要因変わらずで、試料中の馬の肉は、その後の傾斜牛肉としてミオグロビンで二倍の豊富だった場合<html>行は、1よりも大きいであろう。 <img alt="図1" src="/files/ftp_upload/54420/54420fig1.jpg" /> 牛肉で馬w / wの1％の保持時間に対する 図1. MRM遷移強度を。遷移はそれぞれ、黒、青と緑、オレンジ色で示され、752→（1269、706、248、1366）です。マーカーペプチドはHPGDFGADAQGAMTKです。 4遷移フラグメントはここで、yはそれぞれ、Y 13、Y 7、Y 2及びY 14で表さNペプチドのC末端からN個のアミノ酸で計数表すことができます。信号対雑音比は、4つの遷移上で23から53まで変化します。追加の赤い線は0％馬、比較のために100％ビーフのための752→1269推移を示しています。保持時間の唯一の非ゼロの領域が表示されます。この図は、ワトソンらから変更されている。3。エス/ ftp_upload / 54420 / 54420fig1large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/54420/54420fig2.jpg" /> パーセント遷移ピーク面積として牛肉で馬対重量のパーセント重量などの牛肉で馬の 図2. プロット、。プロットは、ペプチド牛肉（767）と馬（752）との両方のためのyの13フラグメントイオンのペアを使用しています。 Aはピーク面積を表す場合には、縦軸が100 A、H /（H + B）です。最良適合線（R 2 = 0.99）の傾きは1.03です。この図は、ワトソンら 3から変更されている。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54420/54420fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication at JoVE.com

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

肉認証にペプチド応用のMRM質量分析法を用いて混合物中の種の決意と定量

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

species-determination-quantitation-mixtures-using-mrm-mass

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.5K ビュー.  Institute of Food Research. We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

ビデオ: Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

液体クロマトグラフィー/質量分析法による有毒フグの改善されたポリメラーゼ連鎖反応 - 制限断片長多型ジェノタイピング

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

遺伝学

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

安定同位体標識するための還元ジメチル化を用いた定量的プロテオミクス

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

化学

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

プロテオミクスから骨格筋の性別二型

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

医学

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液体クロマトグラフィーによるイオン移動 QTOF 質量分析法によるタンパク質のバイオ医薬品の宿主細胞蛋白質の定量化のための HS MRM アッセイ

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

質量分析に基づくプロテオミクスアプローチによるグローバルで信頼性の高いタンパク質R-メチル化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

生化学

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

ペプチド学研究におけるアミンの還元メチル化を用いたサンプル調製と相対定量

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

生物学

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

選択した組織領域を用いた質量分析ベースのプロテオミクスのための合理化されたアプローチ

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

採用プロテアーゼ及び酸触媒ラベリングワークフロー<sup&gt; 18</sup&gt; O-濃縮水

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...