この作品は、全米科学財団（MCB 1021831、JM）とJ.＆E.生物医学研究プログラム（保健フロリダ部、JM）によってサポートされていました。

1。試料調製<ol><li>サンプル調製、不要なアンケージングを防ぐために、暗室内のすべてのサンプル操作を行ってください。 </li><li> 50 mMのHEPESでN、N、N &#39;、N&#39; -テトラキス[（オキシカルボニル）メチル] -1,2 -エタンジアミン） - （2 -ニトロ-4,5 -ジメトキシフェニル） - DM-nitrophen（（1を可溶化バッファー、100mMのKCl、400μMの最終濃度pHは7.0（ε350nmで = 4330 M -1 cm -1の9）。 </li><li> [DM-nitrophen]：の[Ca 2 +]の望ましい比率を達成するために、0.1M原液からのCaCl 2を追加します。 10μMを超えるCa 2 +の関連についてのK dでタンパク質については、の[Ca 2 +]の比1:1の[DM-nitrophen]はアンケージドDM-nitrophenに写真リリースのCa 2 +の結合を防ぐことが望ましい。実際に、カルシウムの90％がタンパク質に結合〜10 nMおよび400 mMのようにするDM-nitrophenおよびCa 2 +の合計濃度であることがDM-nitrophenためのK d値を考慮したK dで=10μMのはapoformになります。一方、K dは 、&lt;10μMのタンパク質への結合のCa 2 +の研究のためには、の[Ca 2 +]低下することが好ましいのとおりでのCa 2 +の錯体形成を防止するために0.95 [DM-nitrophen]比はアポタンパク質ケージ光解離前。 </li><li>参照化合物、K 3 [鉄（III）（CN）6]試料と同じ緩衝液中のNa又は2のCrO 4を可溶化する。 </li></ol> 2。実験のセットアップ<ol><li>基本的な実験構成を図2に示す。 </li><li> 1mmのプローブビームの直径（He-Neレーザーの632nmの出力を、〜5mWのレーザパワー）を調整し、温度が配置セルの中心を通ってプローブ光を伝播させるピンホール（P 2）を使用M 1ミラーを使用して制御セルホルダー。 </li><li>プローブビームを中央にサンプルの後ろにミラー（M 2）を使用します位置敏感型検出器の中心に。 </li><li>検出器の左右側の上部の2つのダイオードと底部つのダイオード間の電圧差ならびに2つのダイオード間の電圧の差があるように検出器の中心にプローブビームを集束ゼロ。 </li><li> 2 355nmのレーザミラーの間に配置され、3mmのピンホール（P 1）を用いてYAGレーザ、FWHM 5ナノ秒）：続いて、ポンプ光、Q-スイッチNdの355nmの出力の直径を形作る。 </li><li> 図2に示されているように、キュベットの中心を通ってポンプビームをCopropagate。これは、両方のレーザビームが測定可能な偏向角を得るために、ほぼ同一直線上に同様に光学セルの中心を通って伝播しPBD信号の振幅、高されていることが重要である。実験条件下で、プローブとポンプビームの交差角が15°未満である。 </li><li>プローブおよびポンプを整列させる参照化合物を使用するより長い時間スケール（〜100ミリ秒）で良好なS / N比や安定したPBD振幅、すなわち 、良好なPBD信号を達成するためにビーム。 </li><li> 355nmのレーザミラーの増分調整することによって、プローブビームに対してポンプビームの位置を調整する。 </li><li>位置検出器上の2つの頂部と底部のフォトダイオードの間の差としてPBD基準信号の振幅を測定する。 PBD信号は速い時間スケール（&lt;10秒）での振幅の急激な増加を示し、 図3で示したように100ミリ秒の時間スケールで安定して維持する必要があります。 PDB振幅の変動に撮影されたシュートは、信号振幅の5％以内であり、信号の再現性は、主に振動によって影響される。 </li><li>吸収された励起レーザパワーオン·アインシュタインの数のPBD信号の線形依存性を、E a を測定することにより放出される熱エネルギーに対してPBD信号振幅の線形性を確認するAは、励起波長での吸光度を基準に相当=（1-10-A）。 </li><li>レーザパワー下記約1,000μJと多光子吸収とそれぞれポンプ光パワーの低下を防止する励起波長が0.5未満での試料/基準化合物の吸光度を維持し、PBD信号の線形性を確保する。 </li></ol> 3。 PBD測定<ol><li>参考のためにPBDトレースの測定を開始します。 1.0センチメートル×1.0センチ1.0センチメートル×0.5センチメートルの石英セルに参照化合物の溶液を配置し、温度制御されたホルダーにセルを配置する。両方の経路長は、同等のPBD振幅を提供する。 </li><li> 3℃の温度増分で16〜35°Cの温度範囲における温度の関数として基準PBD信号を検出</li><li>各々の温度変化の際に、位置検出器上のプローブビームの位置を確認し、再必要に応じて、検出器の中心に位置を調整します。 </li><li> 数2に従って、[（DN / DT）/ C Pは ρ用語の関数としてPBD信号の線形性を確認してください。 </li><li>基準測定のための光学セルの同じ向きを維持参照化合物と同様の光学セル内の試料溶液を置く。 </li><li> PBDは、基準と同じ温度範囲でトレースし、[（のdn / dt）は/ C pが ρ用語に対して試料PBD振幅の線形性を確認する試料を検出する。 </li></ol> 4。データ解析偏向の大きさは、 式1に従って試料加熱（ΔVth）を非熱的及び体積変化（ΔVのnonth）による体積変化に正比例する。 <img src="/files/ftp_upload/50969/50969eq1.jpg" alt="式（1）" /> サンプルの振幅（S）および参照（r）の PBD信号は、それぞれ式2及び図3を用いて説明することができる。 <img src="/files/ftp_upload/50969/50969eq2.jpg" alt="式2" /> <img src="/files/ftp_upload/50969/50969eq3.jpg" alt="式3" /> PBD信号は、計器応答パラメータ（K）に正比例し、アインシュタインの数は、（E a）に吸収される。 式（2）の最初の項は、（DN / DT）（1/ρCp）は 、Qによる溶剤中に放出された熱への信号変化に対応する。のdn / dtの項は、屈折率の温度依存性の変化を表し、ρは溶媒の密度であり、C pは熱容量である。すべてのパラメータは、蒸留水で知られており、蒸留水および適切な緩衝液中の参照化合物についてPBD信号を比較することにより、緩衝液について決定することができる。 Qは熱の量であるreturne溶媒D。 ρ（DN / Dρ）項は、10〜40℃の10の温度範囲で温度に依存しない単位、ほぼ一定である。 Δnは腹筋用語は、溶液中の吸収種の存在のために屈折率の変化に対応し、プローブビームの波長は、溶液中の任意の種の吸収スペクトルに対してずれている場合、それは無視できる程度だ。参照化合物（r）とから生じる信号は、E さhνは光子励起波長におけるエネルギー、E hはν= 355 nmの励起のための80.5キロカロリー/モルである式（3）で表される。 <ol><li> 図3に示されているようにプリトリガ及びポストトリガーPBD信号との間の差としてPBD基準信号の振幅を取る。同様に、高速（A sの 速い ）の振幅を決定し、遅い相（A sの スロー）サンプルPBD信号の。 </li><li>計器応答パラメータを排除するために、Kは、参考のためPBD信号の振幅によって試料PBD信号の振幅をスケーリングする。基準信号サンプル信号の比が式（4）を与え、のように書くことができる。 <img src="/files/ftp_upload/50969/50969eq4.jpg" alt="式4" /> </li><li>この式を使用して、溶液（Q）に放出された熱および[（S / R）Eのプロットのそれぞれ傾きと切片からの光開始反応に伴う非熱体積変化（ΔVのnonth）を決定温度依存的な[（DN / DT）（1/ρCp）]に対するHのν用語。 </li><li>高速と低速のプロセスのための反応体積とエンタルピー変化を決定するために、 式5-7に応じて適切な量子収率が観察され、ボリュームおよびエンタルピー変化をスケール。 <img sRC = &quot;/ files/ftp_upload/50969/50969eq5.jpg&quot; ALT = &quot;式5&quot; /&gt; <img src="/files/ftp_upload/50969/50969eq6.jpg" alt="式6" /> <img src="/files/ftp_upload/50969/50969eq7.jpg" alt="式7" /> 200ミリ秒〜10秒の間の時間スケールで発生する反応速度を有する多段階プロセスについては、反応の個々のステップに関連するボリュームとエンタルピー変化を決定することができる。個々のステップのための振幅および寿命が体積とエンタルピー変化の時間プロファイルを記述する関数F（t）にデータを当てはめることによって分析される。 <img alt="式8" src="/files/ftp_upload/50969/50969eq8.jpg" /> α0は、A、S、 高速に対応しており、 私は、A、S、 低速 、個々のプロセスのために対応してα、τ はどこで個々の反応工程の寿命である。 foの速度定数の温度依存性からrは、個々のプロセスは、活性化エンタルピー及びエントロピーパラメータは容易にアイリングプロットを用いて決定することができる（i = 1 から/τiの k個）。 </li></ol>

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光熱方法の背後にある物理的な原理は、光励起分子は周囲の溶剤1,12の熱加熱で、その結果、基底状態に振動緩和を経由して余分なエネルギーを消費するということです。水などの溶媒の場合、これは急激な体積膨張（ΔVth）を生成する。励起状態の分子はまた、結合開裂/形成および/ ​​または分子の（ファンデ、すなわち変化の分子の大きさを変えることができる分...

このような光音響熱量測定、光熱ビーム偏向（PDB）、およびナノ秒レーザー励起で結合された過渡回折格子などの光熱方法は、短寿命中間体1,2の時間分解研究のための過渡光分光法への強力な代替物である。そのような過渡吸収及びIR分光法などの光学技術とは対照的に、それは周囲の発色団の吸収変化の時間プロファイルを監視、光熱技術は、熱/体積変化の時間依存性を検出し、したがって、光学の時間プロファイルを調べるための貴重なツールである「サイレント」のプロセス。これまでのところ、光音響熱量測定および過渡回折格子が正常グロビン3,4、酸素センサータンパク質とリガンドの相互作用における二原子リガンドの移行を含む光誘起プロセスのコンホメーションのダイナミクスを研究するために適用されているFixL 5、6オキシダーゼ電子とヘム-銅におけるプロトン輸送AND光化学系IIだけでなく、ロドプシン7とクリプトクロム8の立体構造力学の光異性化。 内部発色団および/またはフルオロフォアを欠いている生物学的システムへの光熱技術の適用を拡大するため、PBD技術は依存して、光トリガにケージド化合物を用い、数マイクロ秒以内に、リガンド/基質濃度の増加またはより速いと組み合わせたケージド化合物に関する。このアプローチは、ダイナミクスと内部フルオロフォアまたは発色団を欠いていると商業ストップフロー機器からはアクセスできませんタイムスケール上にあるタンパク質に結合するリガンド/基質に関連する構造変化のエネルギー論のモニタリングを可能にする。ここではダウンニューロンのカルシウムセンサーのC末端ドメインにケージ化合物の熱力学を監視するためのPBDのアプリケーション、Ca 2 +のDM-nitrophen、光切断だけでなく、Ca 2 +の関連についての動態ストリーム調節エレメント拮抗モジュレータ（DREAM）が提示されている。 Ca 2 +を、光にリリース〜300秒の時定数でunphotolysedケージにCa 2 +を 10秒と再バインド内のDM-nitrophenからのものである。一方、apoDREAMの存在下で、ミリ秒の時間スケールで発生する追加の運動を観察し、タンパク質へのリガンド結合を反映している。生物系の高次構造遷移をプローブするPBDの適用は、何らかの形で楽器の難しさのために制限されている、強力かつ再現性のPBD信号を達成するためのプローブとポンプ光の例えば骨の折れる整列。しかし、計測機器のセットアップの綿密な設計、温度の正確な制御、およびプローブとポンプ光の慎重な位置合わせは、広範な上、時間分解体積とエンタルピー変化のモニタリングを可能にする一貫性と堅牢PBD信号を提供10秒で約200ミリ秒の時間スケール。また、modificなど 、同一温度、緩衝液組成、光学セルの向き、レーザパワー、下のサンプルとリファレンストレースの検出を確実にする実験手順の管理ポイントを大幅に測定された反応容量およびエンタルピーでの実験誤差を低減します。

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			<td height="84" style="height:84px;width:216px;">1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N&#39;,N&#39;-Tetraacetic Acid</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">D-6814</td>
			<td style="width:220px;">DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation,</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td style="width:84px;">Sigma Adrich</td>
			<td>0909C</td>
			<td>HEPES buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium ferricyanide(III)</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">702587</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:84px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:220px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chromate</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">307831</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">He-Ne Laser Diode 5mW 635nm </td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">54-179</td>
			<td style="width:220px;">use as a probe beam for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Oscilloscope, </td>
			<td style="width:84px;">LeCroy</td>
			<td style="width:119px;">Wave Surfer 42Xs</td>
			<td style="width:220px;">400 MHz bandwith</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nd:YAG laser</td>
			<td style="width:84px;">Continuum</td>
			<td style="width:119px;">ML II</td>
			<td>pump beam for PBD measurments</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M355; Nd:YAG laser mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">47-324</td>
			<td>laser mirror for 355 nm laser line</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M1 and M2; Laser diode mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">43-532</td>
			<td>visilbe laser flat mirror, wavelength range 300-700 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P1 and P2; Iris Diaphragm</td>
			<td style="width:84px;">Edmind Optics</td>
			<td style="width:119px;">62-649</td>
			<td style="width:220px;">pin hole to shape the probe and pum beams</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">L1; bi-convex lens</td>
			<td style="width:84px;">Thorlabs</td>
			<td style="width:119px;">LB1844</td>
			<td style="width:220px;">a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350 - 2000 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DM, dichroic mirror</td>
			<td style="width:84px;">Thorlab </td>
			<td style="width:119px;">DMLP505</td>
			<td style="width:220px;">a longpass dichroic mirror with a cutoff wavelength of 505 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F1; Edge filter</td>
			<td style="width:84px;">Andower </td>
			<td style="width:119px;">500FH90-25</td>
			<td>a long pass filter with a cutoff wavelength of 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-controlled cuvette holder</td>
			<td style="width:84px;">Quantum Northwest</td>
			<td style="width:119px;">FLASH 300</td>
			<td></td>
		</tr>
	</tbody>
</table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

一緒に光音響熱量測定および熱格子と光熱ビーム偏向は、伝統的なストップを使用してアクセスできない時間スケールをミリ秒、マイクロ秒でのタンパク質の立体構造変化を誘起光の時間プロファイルボリュームとエンタルピー変化を監視する光熱方法のファミリーに属するフローの楽器。さらに、体積および/またはエンタルピーの全体的な変化がプローブされているので、これらの技術は、タンパク質およびフルオロフォアおよび発色団又はラベルを欠く他の生体高分子に適用することができる。カルシウムトランスデューサ、このような神経細胞のカルシウムセンサー、ケージカルシウム化合物、DM-nitrophenへの結合のCa 2 +に関連した構造変化のダイナミクスとエネルギー論を監視するには、フォトトリガーに遊離カルシウムの急速な（τ&lt;20秒）増加して採用されている濃度及び関連するボリュームとエンタルピー変化は、光熱ビーム偏向技術を用いてプローブする。

PBDの代表的な例は、Ca 2 + DM-nitrophenからのCa 2 +放出光は、図3に示されているトレース。遅い位相がCA 2 + nonphotolysedケージとの結合を反映しているのに対し、FASTフェーズは、Ca 2 +のDM は 、nitrophenおよびCa 2 +の解放の光切断に対応しています。温度依存係数[C のpρ/（のdn / dt）が〕の関数として基準化合物の大きさにスケーリ...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection | Chemistry | Video

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

ここでは、下流の調節エレメント拮抗モジュレータ、マイクロ秒およびミリ秒ダイナミクスおよびニューロンカルシウムセンサーへのカルシウムアソシエーションに関連付けられている構造変化のエネルギー論をモニターするために、DM-nitrophen、ケージドカルシウム化合物と組み合わせて光熱ビーム偏向技術の適用を報告。

光熱ビーム偏向によって分離したタンパク質におけるミリ秒以下構造変化

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

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8.9K ビュー.  Florida International University. ここでは、下流の調節エレメント拮抗モジュレータ、マイクロ秒およびミリ秒ダイナミクスおよびニューロンカルシウムセンサーへのカルシウムアソシエーションに関連付けられている構造変化のエネルギー論をモニターするために、DM-nitrophen、ケージドカルシウム化合物と組み合わせて光熱ビーム偏向技術の適用を報告。 

ビデオ: Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

蛍光ベースの氷面アフィニティーによる不凍タンパク質の氷結合平面の決定

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

化学

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

妊娠中にラット肝組織中の脂肪酸の組成変化を分析するために、ガスクロマトグラフィーの使用

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

高速液体クロマトグラフィー結合フローシンチレーションにより放射性標識およびホスホイノシチドの細胞レベルの定量化

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

サルヴィとTOF-SIMSによる水中で水和タンパク質のその場キャラクタリゼーションで

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

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 ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

時間分解光物性評価のトリプレット収穫有機化合物無酸素環境使用、iCCD カメラ

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

バインドの動的・電気泳動光散乱による解析 DNA 磁性粒子

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

植物の効率的な方法で複数のキメラ蛍光融合タンパク質の共発現

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

F− 310 Nm 剥離によって示されている陰イオンの光電子イメージング

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

微結晶の時間分解フェムト秒シリアル結晶学 x 線レーザーのための高粘度押出を改善

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...