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Kit\u3092\u4f7f\u3046\u3053\u3068\u3067\u3001\uff08\u9ad8pH\u9006\u76f8\uff09\u5206\u753b\u2192\uff08\u4f4epH\u9006\u76f8\uff09\u5206\u753b\u2192MS\u3068\u3044\u3046\u6d41\u308c\u3068\u306a\u308a\u3001\u7570\u306a\u308b\u5206\u753b\u6cd5\u30922\u3064\u9023\u7d9a\u3055\u305b\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u308c\u306b\u3088\u308a\u3001\u30b5\u30f3\u30d7\u30eb\u306e\u8907\u96d1\u6027\u3092\u4f4e\u6e1b\u3055\u305b\u3001\u540c\u5b9a\u6570\u306e\u5411\u4e0a\u304c\u671f\u5f85\u3067\u304d\u307e\u3059\u3002\r\n\r\n\r\n\u56f33\u306b\u3001Pierce High pH Reversed-Phase Peptide Fractionation 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Fractionation Kit\u3067\u5206\u753b\u3057\u305f\u5834\u5408\uff08\u8d64\u3044\u30d0\u30fc\uff09\u3001\u30da\u30d7\u30c1\u30c9\u540c\u5b9a\u657053,024\u3001\u30bf\u30f3\u30d1\u30af\u8cea\u540c\u5b9a\u65707,477\u3068\u30012\u500d\u7a0b\u5ea6\u306e\u540c\u5b9a\u6570\u5411\u4e0a\u306b\u6210\u529f\u3057\u307e\u3057\u305f\u3002\r\nPierce High pH \u2026","hashtag":"ThermoFisherJP","subscribe":"content","subscribe_url":"","activestatus":"1","singular":"1","twitter_popup":"1","refresh":"0","nonce":"01c3793ea9","postid":"18504","servertime":"1732387891","ajaxurl":"https:\/\/www.thermofisher.com\/blog\/wp-admin\/admin-ajax.php"}; /* ]]> */ </script> <script type="text/javascript" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/plugins/mashsharer/assets/js/mashsb.min.js?ver=4.0.47" id="mashsb-js"></script> <script type="text/javascript" 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Scientific\u2122 Pierce\u2122 High pH Reversed-Phase Peptide Fractionation Kit\u306f\u3001\u9ad8pH\u9006\u76f8\u30af\u30ed\u30de\u30c8\u30b0\u30e9\u30d5\u30a3\u30fc\u3067\u30da\u30d7\u30c1\u30c9\u3092\u5206\u753b\u3059\u308b\u88fd\u54c1\u3067\u3059\u3002LC-MS\u306eLC\u3067\u306f\u3001\u4f4epH\u306e\u9006\u76f8\u30af\u30ed\u30de\u30c8\u30b0\u30e9\u30d5\u30a3\u30fc\u304c\u4f7f\u308f\u308c\u307e\u3059\u3002\u305d\u306e\u305f\u3081\u3001Pierce High pH Reversed-Phase Peptide Fractionation Kit\u3092\u4f7f\u3046\u3053\u3068\u3067\u3001\uff08\u9ad8pH\u9006\u76f8\uff09\u5206\u753b\u2192\uff08\u4f4epH\u9006\u76f8\uff09\u5206\u753b\u2192MS\u3068\u3044\u3046\u6d41\u308c\u3068\u306a\u308a\u3001\u7570\u306a\u308b\u5206\u753b\u6cd5\u30922\u3064\u9023\u7d9a\u3055\u305b\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u308c\u306b\u3088\u308a\u3001\u30b5\u30f3\u30d7\u30eb\u306e\u8907\u96d1\u6027\u3092\u4f4e\u6e1b\u3055\u305b\u3001\u540c\u5b9a\u6570\u306e\u5411\u4e0a\u304c\u671f\u5f85\u3067\u304d\u307e\u3059\u3002\r\n\r\n\r\n\u56f33\u306b\u3001Pierce High pH Reversed-Phase Peptide Fractionation 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High pH Reversed-Phase Peptide Fractionation Kit\u306b\u306f\u30e1\u30ea\u30c3\u30c8\u304c\u3042\u308b\u3068\u8a00\u3048\u308b\u3067\u3057\u3087\u3046\u3002\r\n\u3067\u306f\u3001\u809d\u5fc3\u306e\u540c\u5b9a\u6570\u306f\u3069\u3046\u3067\u3057\u3087\u3046\u304b\u3002\u56f34\u3092\u898b\u3066\u307f\u307e\u3057\u3087\u3046\u3002\r\n\r\n\r\n\u56f34\u3067\u306f\u3001\u3053\u306e\u30ad\u30c3\u30c8\u3067\u5206\u753b\u3057\u305f\u30b5\u30f3\u30d7\u30eb\u3068\u5206\u753b\u3057\u3066\u3044\u306a\u3044\u30b5\u30f3\u30d7\u30eb\u306e\u30da\u30d7\u30c1\u30c9\uff08A\uff09\u3068\u30bf\u30f3\u30d1\u30af\u8cea\uff08B\uff09\u306e\u540c\u5b9a\u6570\u3092\u305d\u308c\u305e\u308c\u6bd4\u8f03\u3057\u3066\u3044\u307e\u3059\u3002\u672a\u5206\u753b\u306e\u5834\u5408\uff08\u30b0\u30ec\u30fc\u306e\u30d0\u30fc\uff09\u3001\u30da\u30d7\u30c1\u30c9\u540c\u5b9a\u657022,487\u3001\u30bf\u30f3\u30d1\u30af\u8cea\u540c\u5b9a\u65704,444\u3060\u3063\u305f\u306e\u306b\u5bfe\u3057\u3001Pierce High pH Reversed-Phase Peptide Fractionation Kit\u3067\u5206\u753b\u3057\u305f\u5834\u5408\uff08\u8d64\u3044\u30d0\u30fc\uff09\u3001\u30da\u30d7\u30c1\u30c9\u540c\u5b9a\u657053,024\u3001\u30bf\u30f3\u30d1\u30af\u8cea\u540c\u5b9a\u65707,477\u3068\u30012\u500d\u7a0b\u5ea6\u306e\u540c\u5b9a\u6570\u5411\u4e0a\u306b\u6210\u529f\u3057\u307e\u3057\u305f\u3002\r\nPierce High pH \u2026"}; /* ]]> */ </script> <script type="text/javascript" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/plugins/mashshare-networks/assets/js/mashnet.min.js?ver=2.5.3" id="mashnet-js"></script> <script type="text/javascript" src="https://www.thermofisher.com/blog//wp-includes/js/underscore.min.js?ver=1.13.4" id="underscore-js"></script> <script type="text/javascript" src="https://www.thermofisher.com/blog//wp-includes/js/backbone.min.js?ver=1.5.0" id="backbone-js"></script> <script type="text/javascript" id="wp-api-js-js-extra"> /* <![CDATA[ */ var WP_API_Settings = 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href="https://www.thermofisher.com/blog/learning-at-the-bench/contact-us/"><span >お問い合わせ</span></a></li> </ul></nav></div></section> </div><div class="breadcrumb"><span class="breadcrumb-link-wrap"><a href="https://www.thermofisher.com/blog/?">Accelerating Science</a></span><span class="breadcrumb-link-wrap"><a class="breadcrumb-link" href="https://www.thermofisher.com/blog/learning-at-the-bench/"><span class="breadcrumb-link-text-wrap">Learning at the Bench</span></a><meta ></span> <span aria-label="breadcrumb separator">/</span> <span class="breadcrumb-link-wrap"><a class="breadcrumb-link" href="https://www.thermofisher.com/blog/learning-at-the-bench/analysis/"><span class="breadcrumb-link-text-wrap">分析・測定関連</span></a><meta ></span> <span aria-label="breadcrumb separator">/</span> <span class="breadcrumb-current-title">プロテオーム解析のサンプル調製：LC-MS前の分画による同定数向上</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18504 post type-post status-publish format-standard has-post-thumbnail category-analysis tag-protein-analysis tag-mass-spectrometry entry" aria-label="プロテオーム解析のサンプル調製：LC-MS前の分画による同定数向上"><header class="entry-header"><h1 class="entry-title">プロテオーム解析のサンプル調製：LC-MS前の分画による同定数向上</h1> <span class="post-info-author"> 作成者 <a href="https://www.thermofisher.com/blog/author/lgbadmin/" title="LatB Staff の投稿" rel="author">LatB Staff</a> </span> <span class="post-info-date"> 11.13.2024 </span> </header><div class="entry-content"><p><img decoding="async" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/06/48927600915c949e1277a7b63c505227.jpg" alt="" width="1000" height="563" class="aligncenter size-full wp-image-17387" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/06/48927600915c949e1277a7b63c505227.jpg 1000w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/06/48927600915c949e1277a7b63c505227-300x169.jpg 300w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/06/48927600915c949e1277a7b63c505227-768x432.jpg 768w" sizes="(max-width: 1000px) 100vw, 1000px" /><br /> プロテオーム解析、つまり生体内に存在するタンパク質の網羅的な解析が実施されるようになり30年以上が経とうとしています。この間、LC-MSの機器や技術は発展を繰り返してきましたが、いまだに、「細胞中の全タンパク質を解析すること」は不可能です。<br /> これは、「ダイナミックレンジ」を考えればおのずと明らかです。生物学的サンプル中のタンパク質濃度は、ダイナミックレンジが10桁を超えると言われています。つまり、量の少ないタンパク質は、量の多いタンパク質の1/10<sup>10</sup>しか存在しないのです。一方、質量分析計（MS）のダイナミックレンジは、高性能の機器でも5桁程度が限界。つまり、「網羅的」にはまだまだ遠いのです。<br /> これを解決するために、質量分析前に高速液体クロマトグラフィー（LC）で分画するLC-MSが実施されています。プロテオーム解析において、現在主流の方法です。もちろんMSのみよりも、MS前にLCを実施することで同定数は増えますが、それでも10桁のダイナミックレンジはカバーしきれません。<br /> そんな中で、せめて少しでも同定数を増やすことに焦点をおいて、このブログではLC-MS前、つまりサンプル調製時の分画についてご紹介します。</p> <div id="toc_container" class="no_bullets"><p class="toc_title">▼もくじ</p><ul class="toc_list"><li><a href="#pH">高pH逆相レジンによるペプチド分画</a></li><li><a href="#i">その他の分画方法</a></li><li><a href="#i-2">まとめ</a><ul><li><a href="#i-3">質量分析前処理ハンドブック～プロテオーム解析に必須な試薬やツール</a></li></ul></li></ul></div> <h2><span id="pH">高pH逆相レジンによるペプチド分画</span></h2> <p>まず、図1はLC-MS前のサンプル調製の一般的な流れです。<br /> <div id="attachment_18507" style="width: 776px" class="wp-caption aligncenter"><img decoding="async" aria-describedby="caption-attachment-18507" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/af3672546e3568e85f80559acf2b71e4.jpg" alt="図1. LC-MSのサンプル前処理の一般的な流れ" width="766" height="92" class="size-full wp-image-18507" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/af3672546e3568e85f80559acf2b71e4.jpg 766w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/af3672546e3568e85f80559acf2b71e4-300x36.jpg 300w" sizes="(max-width: 766px) 100vw, 766px" /><p id="caption-attachment-18507" class="wp-caption-text">図1. LC-MSのサンプル前処理の一般的な流れ</p></div></p> <p>では「サンプル調製時の分画」はどこで実施するのでしょうか。<br /> いくつかのアプローチがありますが、まずは、消化後のペプチドを分画する方法をご紹介します。この方法では、LC-MS直前のクリーンアップステップを「分画」に変更します（図2）。<br /> <div id="attachment_18508" style="width: 770px" class="wp-caption aligncenter"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-18508" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/71ef1567b21143e7bfbf17177289edea.jpg" alt="図2. LC-MS前分画の例" width="760" height="82" class="size-full wp-image-18508" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/71ef1567b21143e7bfbf17177289edea.jpg 760w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/71ef1567b21143e7bfbf17177289edea-300x32.jpg 300w" sizes="(max-width: 760px) 100vw, 760px" /><p id="caption-attachment-18508" class="wp-caption-text">図2. LC-MS前分画の例</p></div></p> <p>ここで注意しておきたいのは、LC-MSの「LC」も分画のひとつであるという点です。つまり、上図の流れは、酵素消化の後、<font color="red"><b>分画→分画→MS</b></font>と進むことになります。そのため、この2つの連続するステップでは、異なる分画をしないと高い効果が望めません。<br /> Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/84868?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">Pierce™ High pH Reversed-Phase Peptide Fractionation Kit</a>は、高pH逆相クロマトグラフィーでペプチドを分画する製品です。LC-MSのLCでは、低pHの逆相クロマトグラフィーが使われます。そのため、Pierce High pH Reversed-Phase Peptide Fractionation Kitを使うことで、<font color="red"><b>（高pH逆相）分画→（低pH逆相）分画→MS</b></font>という流れとなり、異なる分画法を2つ連続させることができます。これにより、サンプルの複雑性を低減させ、同定数の向上が期待できます。<br /> <div id="attachment_18509" style="width: 633px" class="wp-caption aligncenter"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-18509" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/ab04e5237e121f6314a84440c474b35d.jpg" alt="図3. High pH Reversed-Phase Peptide Fractionation Kitの流れ" width="623" height="568" class="size-full wp-image-18509" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/ab04e5237e121f6314a84440c474b35d.jpg 623w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/ab04e5237e121f6314a84440c474b35d-300x274.jpg 300w" sizes="(max-width: 623px) 100vw, 623px" /><p id="caption-attachment-18509" class="wp-caption-text">図3. High pH Reversed-Phase Peptide Fractionation Kitの流れ</p></div></p> <p>図3に、Pierce High pH Reversed-Phase Peptide Fractionation Kitのキットの流れを示しました。まずカラムを平衡化し、消化ペプチドを添加して洗浄した後、アセトニトリルの濃度を変えて8つの画分に溶出します。それぞれをLC-MSで解析するという流れです。特別な機器は必要なく、スピンカラムで気軽に分画できるのがメリットのひとつです。<br /> 各バッファーにはトリエチルアミンが含まれており、高pHが維持された状態で実施されます。また、一般的なLC-MSサンプル前処理におけるクリーンナップでもよく逆相カラムが使用されるように、このキットも原理は逆相カラムですので、工程の中でバッファー成分や塩成分が除去されます。よってキット処理後のクリーンアップが必要ありません。LC-MS前のペプチド分画法としてSCX（陽イオンクロマトグラフィー）が使われることもありますが、SCXの場合、分画後に脱塩が必要なため、その点でもPierce High pH Reversed-Phase Peptide Fractionation Kitにはメリットがあると言えるでしょう。<br /> では、肝心の同定数はどうでしょうか。図4を見てみましょう。<br /> <div id="attachment_18510" style="width: 870px" class="wp-caption aligncenter"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-18510" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/878690a0eb50a2e226c995b82e8647e5.jpg" alt="図4. 分画サンプルと未分画サンプルのプロファイル HeLa細胞をトリプシンで消化し、Pierce High pH Reversed-Phase Peptide Fractionation Kitで分画したものと分画していないものを、それぞれThermo Scientific™ Orbitrap Fusion™ Tribrid™ 質量分析計にて分析しました A：ペプチド同定数、B：タンパク質同定数" width="860" height="493" class="size-full wp-image-18510" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/878690a0eb50a2e226c995b82e8647e5.jpg 860w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/878690a0eb50a2e226c995b82e8647e5-300x172.jpg 300w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/878690a0eb50a2e226c995b82e8647e5-768x440.jpg 768w" sizes="(max-width: 860px) 100vw, 860px" /><p id="caption-attachment-18510" class="wp-caption-text">図4. 分画サンプルと未分画サンプルのプロファイル<br />HeLa細胞をトリプシンで消化し、Pierce High pH Reversed-Phase Peptide Fractionation Kitで分画したものと分画していないものを、それぞれThermo Scientific™ Orbitrap Fusion™ Tribrid™ 質量分析計にて分析しました<br />A：ペプチド同定数、B：タンパク質同定数</p></div></p> <p>図4では、このキットで分画したサンプルと分画していないサンプルのペプチド（A）とタンパク質（B）の同定数をそれぞれ比較しています。未分画の場合（グレーのバー）、ペプチド同定数22,487、タンパク質同定数4,444だったのに対し、Pierce High pH Reversed-Phase Peptide Fractionation Kitで分画した場合（赤いバー）、ペプチド同定数53,024、タンパク質同定数7,477と、2倍程度の同定数向上に成功しました。<br /> Pierce High pH Reversed-Phase Peptide Fractionation Kitに関するWhite Paperは<a href="https://tools.thermofisher.com/content/sfs/brochures/PS-high-pH-RP-Kit-White-Paper.pdf?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">こちら</a>です。</p> <h2><span id="i">その他の分画方法</span></h2> <p>では、LC-MS前に分画する方法として、上述以外にどんな方法があるでしょうか。<br /> 他には、タンパク質を抽出する段階で「分画抽出」する方法があります（図5）。<br /> <div id="attachment_18511" style="width: 776px" class="wp-caption aligncenter"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-18511" src="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/d31138a7f44f71753478307e5c3b648d.jpg" alt="図5. LC-MS前分画の例" width="766" height="92" class="size-full wp-image-18511" srcset="https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/d31138a7f44f71753478307e5c3b648d.jpg 766w, https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/11/d31138a7f44f71753478307e5c3b648d-300x36.jpg 300w" sizes="(max-width: 766px) 100vw, 766px" /><p id="caption-attachment-18511" class="wp-caption-text">図5. LC-MS前分画の例</p></div></p> <p>例えば核タンパク質と細胞質タンパク質を別々に分画抽出（Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/jp/ja/78833?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">NE-PER™ Nuclear and Cytoplasmic Extraction Reagents</a>）することで、全タンパク質を抽出した場合よりも、タンパク質の同定数を増やすことに成功しています（データは<a href="https://www.thermofisher.com/blog/learning-at-the-bench/protein-fractionation-and-concentration-for-ms/#MS?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">こちら</a>）。<br /> また、膜タンパク質は水溶性タンパク質よりも抽出が難しいため、全タンパク質を抽出する試薬では抽出効率が低いことが多いです。そのため、LC-MS解析時の膜タンパク質同定数も低くなることがあります。このような場合、膜タンパク質抽出用のキット（例えばThermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/89842?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">Mem-PER™ Plus Membrane Protein Extraction Kit</a>）で分画抽出することで、膜タンパク質の同定数を上げることができます（表1）。</p> <table> <caption><b>表1. HEK292細胞から、全タンパク質抽出用のM-PERと膜タンパク質分画抽出用のMem-PERを用いて抽出<br />それぞれのサンプルを、Velos Pro Orbitrap質量分析計で分析、タンパク質同定数を比較</b></caption> <tbody> <tr bgcolor="Teal"> <td style="text-align: center" width="300"><font color="white"><b> Reagent</b></font></td> <td style="text-align: center" width="175"><font color="white"><b>M-PER</b></font></td> <td style="text-align: center" width="175"><font color="white"><b>Mem-PER Plus</b></font></td> </tr> <tr> <td style="text-align: left" width="300">Total number of proteins identified</td> <td style="text-align: center" width="175">451</td> <td style="text-align: center" width="175">425</td> </tr> <tr> <td style="text-align: left" width="300">Integral membrane proteins identified</td> <td style="text-align: center" width="175">30</td> <td style="text-align: center" width="175">90</td> </tr> <tr> <td style="text-align: left" width="300">Integral membrane proteins identified,<br />percent of total</td> <td style="text-align: center" width="175">6.7</td> <td style="text-align: center" width="175">21.2</td> </tr> </tbody> </table> <p>表1は、全タンパク質抽出用のM-PERと、膜タンパク質分画抽出用のMem-PERを用いて分画抽出し、それぞれをLC-MSで分析した結果です。M-PERでは、膜タンパク質同定数が僅か30だったのに対し、Mem-PERでは90の膜タンパク質同定に成功しています。ただし、Mem-PERは膜タンパク質の抽出に特化しているため、トータルタンパク質同定数ではM-PERでは451、Mem-PERでは425と、Mem-PERの方が同定数は低い結果となりました。そのため、解析対象や目的によって抽出試薬によって使い分けることをおすすめします。<br /> Mem-PER以外の、サンプルから分画抽出できる試薬やキットを確認されたい方は<a href="https://www.thermofisher.com/jp/ja/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/subcellular-fractionation.html?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">こちら</a>をご覧ください。</p> <p>最後に、血清や血漿サンプルの分画についてお話しておきましょう。血液サンプルの場合、「分画」というよりも「除去」になりますが、サンプル中の一部のタンパク質を取り出して同定数を増やすという点では同じです。<br /> 血清や血漿がサンプルの場合、LC-MSでとにかく邪魔なのは、サンプル中に大量に含まれるアルブミンや抗体です。星は夜にしか見えません。しかし本当は、昼間も空の上で星が輝いています。昼間に星が見えないのは、太陽という圧倒的な光量を誇る恒星がいるからです。血液サンプルも同じ。血液サンプルにおけるアルブミンやIgGは太陽、見えない星たちはアルブミンやIgGほど存在量が多くないタンパク質たちです。星たちを見るために、太陽を除く作業を行いましょう。<br /> 表2に、血液サンプル向けのアルブミンや抗体の除去キットを掲載しました。アルブミンや抗体以外にも豊富に含まれるタンパク質を除けるキットもあり、便利です。状況に応じて選択しましょう。</p> <table> <caption><b>表2. 血液サンプル中の豊富に含まれるタンパク質を除去するキット一覧</b></caption> <tbody> <tr bgcolor="gray"> <td style="text-align: center" width="200"><font color="white"><b> 製品名</b></font></td> <td style="text-align: center" width="150"><font color="white"><b>生物種</b></font></td> <td style="text-align: center" width="350"><font color="white"><b>除去されるもの</b></font></td> </tr> <tr> <td style="text-align: left" width="200">Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/A36370?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">High Select™ Top14 Abundant Protein Depletion Columns</a></td> <td style="text-align: left" width="150">Human</td> <td style="text-align: left" width="350">Albumin, IgG, IgA, IgM, IgD, IgE, IgG(light chain), α1-Acid glycoprotein, Fibrinogen, Haptoglobin,α1- Antitrypsin, α2-Marcoglobulin, Transferin, Apolipoprotein A-I</td> </tr> <tr> <td style="text-align: left" width="200">Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/A36365?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">HSA/Immunoglobulin Depletion Columns</a></td> <td style="text-align: left" width="150">Human</td> <td style="text-align: left" width="350">Albumin, IgG, IgA, IgM, IgD, IgE, IgG(light chain)</td> </tr> <tr> <td style="text-align: left" width="200">Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/89875?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">Pierce™ Albumin/IgG Removal Ki</a></td> <td style="text-align: left" width="150">Human, Monkey,<br />Swine, Rabbit</td> <td style="text-align: left" width="350">Albumin, IgG</td> </tr> <tr> <td style="text-align: left" width="200">Thermo Scientific™ <a href="https://www.thermofisher.com/order/catalog/product/85160?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">Pierce™ Albumin Depletion Kit</a></td> <td style="text-align: left" width="150">Human, Swine,<br />Sheep, Rabbit</td> <td style="text-align: left" width="350">Albumin</td> </tr> </tbody> </table> <h2><span id="i-2">まとめ</span></h2> <p>LC-MSの技術は発達の一途を辿ってきましたが、サンプル中のタンパク質をすべて同定することはまだまだできません。しかし、サンプルに応じた分画を適切なタイミングで実施することで、同定数を増やすことができます。</p> <p>プロテオーム解析に最適なThermo Scientific™ Orbitrap™質量分析計は<a href="https://www.thermofisher.com/jp/ja/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms.html?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">こちら</a><br /> プロテオミクス基礎シリーズe-learningは<a href="https://www.thermofisher.com/jp/ja/home/technical-resources/e-learnings/lc-ms.html?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">こちら</a></p> <h3><span id="i-3">質量分析前処理ハンドブック～プロテオーム解析に必須な試薬やツール</span></h3> <p><a class="btn btn-red" href="https://resource.thermofisher.com/page/WE41623/documents/COL32447-Mass-Spec-Essentials-Handbook-Global-FLR.pdf?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" target="_blank" rel="noopener noreferrer">ダウンロードはこちら</a></p> <p>このブログを読んだ人はこんなブログも読んでいます：<br /> <a href="https://www.thermofisher.com/blog/learning-at-the-bench/protein-analysis-mass-spectrometry-bid-ts-mbr-24035/?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">プロテオーム解析のサンプル調製、おさえておきたいポイント</a><br /> <a href="https://www.thermofisher.com/blog/learning-at-the-bench/protein-analysis-mass-spectrometry-bid-ts-mbr-24065/?CID=bid_cbu_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_2_bid_ts_mbr_24078_Social_LAB" rel="noopener" target="_blank">リン酸化プロテオミクスのLC-MSサンプル調製、おさえておきたいポイント</a><br /> <a 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