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

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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 border="0" cellpadding="0" cellspacing="0" width="1076">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Uniprot database</td>
			<td style="width:243px;">www.uniprot.org</td>
			<td style="width:119px;"></td>
			<td style="width:499px;">Freely accessible database of protein sequences</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Skyline software</td>
			<td style="width:243px;">www.skyline.gs.washington.edu</td>
			<td style="width:119px;"></td>
			<td style="width:499px;">Free software to download that enables the creation of targeted methods for proteomic studies, peptide and fragment prediction&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium bicarbonate</td>
			<td style="width:243px;">Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td>
			<td style="width:119px;">O9830</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Methanol, HPLC grade</td>
			<td style="width:243px;">Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td>
			<td style="width:119px;">10674922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acetonitrile, HPLC grade</td>
			<td style="width:243px;">Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td>
			<td style="width:119px;">10010010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Urea</td>
			<td style="width:243px;">Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td>
			<td style="width:119px;">U5378</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin(from bovine pancreas treated with TPCK)</td>
			<td style="width:243px;">Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td>
			<td style="width:119px;">T1426</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formic acid&#160;</td>
			<td style="width:243px;">Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coomassie Plus Protein Assay Reagent</td>
			<td style="width:243px;">Thermo Fisher Scientific www.thermofisher.com</td>
			<td style="width:119px;">1856210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protein standard</td>
			<td style="width:243px;">Sigma-Aldrich Co Ltd, Gillingham, UK www.sigmaaldrich.com</td>
			<td style="width:119px;">P0914</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra Turrax homogeniser T25</td>
			<td style="width:243px;">Fisher Scientific, Loughoborough, UK www. fisher.co.uk</td>
			<td style="width:119px;">13190693</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Edmund and Buhler KS10 lab shaker</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heraeus Fresco 17 Centrifuge</td>
			<td style="width:243px;">Thermo Fisher Scientific www.thermoscientific.com</td>
			<td style="width:119px;">75002420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum centrifuge RC 1022</td>
			<td style="width:243px;">Jouan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plate Reader</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Strata-X 33u polymeric reversed-phase cartridges 60 mg/3 ml tubes</td>
			<td style="width:243px;">Phenomenex, Macclesfield, UK</td>
			<td style="width:119px;">8B-S100-UBJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4000 QTrap triple-quadrupole mass spectrometer</td>
			<td style="width:243px;">AB Sciex, Warrington, UK www.sciex.com</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1200 rapid resolution LC system</td>
			<td style="width:243px;">Agilent, Stockport, UK</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">XB C18 reversed-phase capillary column (100 x 2.1mm, 2.6&#181; particle size)</td>
			<td style="width:243px;">Phenomenex, Macclesfield, UK www.phenomenex.com</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Analyst 1.6.2 software</td>
			<td style="width:243px;">AB Sciex, Warrington, UK www.sciex.com</td>
			<td style="width:119px;"></td>
			<td>QTrap data acquisition and analysis, including peak area integration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autosampler vials</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></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/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

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

生化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

ブルーネイティブPAGEおよびタンパク質相関プロファイリングによってアフィニティー精製タンパク質複合体の解決

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

定量的質量分析のための脂肪滴の分離

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

四バイオセンサープラットフォームを用いた高親和性抗体の抗原結合速度の決意

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

質量分析を用いた識別 RNA 結合領域のための試料作製

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI-TOF 質量分析法によるマウス組織におけるプロテアーゼの特異性の定量抽出: 特異性変化を引き起こすに PH を操作します。

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

校正と単一のヌクレオチドの拡張子と MALDI-TOF 質量分析を用いた DNA 修復アッセイ

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

アンジオテンシンIIによるニトロペプチドプロファイリングと同定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

質量光測定による抗体-抗原アフィニティーの迅速な測定

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...