This work was supported by NSF/DBI-1255991 grant to IML.

모세관 마이크로 반응기 1. 준비 <ol><li> 100 μm의 내경 (ID)이 360 ㎛의 외경 (OD) 7-8cm 길이로 모세관 유리 모세관 칼 3-5 센티미터 20 μm의 ID X 0.09의 OD 모세관 X 컷; 어떤 돌출 버없이, 모두 모세관 끝이 깨끗하고 스트레이트 컷을 현미경으로 확인합니다. </li><li> ~ 6mm의 길이, 100 μm의 ID X 360 μm의 외경 모세관의 한쪽 끝으로 20 μm의 ID X 90 μm의 외경 모세관을 삽입; 필요한 경우에, 현미경이 동작을 관찰한다. </li><li> 면봉의 끝이 90 μm의 OD / 100 μm의 아이디 모세 혈관의 교차점 주위에 접착제 E6000의 작은 방울을 적용하고 RT에서 접착제 경화 O / N을 할 수 있습니다. </li><li> ~ 10-15mm의 길이에 삽입 된 20 μm의 ID X 90 μm의 외경 모세관을 잘라; 이것은 전기 분무 이온화 방출 될 것입니다. </li><li> 미리 잘라 1/32 &quot;OD의 PEEK, 1/16&quot;OD의 PEEK 및 1/16 &quot;OD의 PTFE 욕조길이 4-5cm의 조각 보내고; 이러한 누출이없는 연결을 제공하기 위해 모세관 조합에 사용되는 소매 될 것입니다. </li><li> 까꿍 조합에 100 μm의 ID X 360 μm의 외경 모세관의 반대쪽 끝을 연결합니다; 누출이없는 연결을 제공하기위한 1/32 &quot;PEEK 슬리브를 사용합니다. </li><li> 삽입 / 노조의 반대쪽에 5cm 길이 1/16 &quot;PTFE 튜브 ~의 조각을 조입니다. </li><li> 사전 세척 / 2 ㎖의 유리 병을 건조에 C18 (5 μm의) 입자 ~ 4 mg의 무게; 0.5 ml의 이소프로판올을 추가 바이알을 닫고 초음파 목욕 슬러리를 분산. </li><li> , 250 μL 주사기 약 200 ㎕의 슬러리를 철수 PEEK 조합의 1/16 &quot;PTFE 튜브에서 주사기 바늘을 삽입하고 천천히 100 μm의 ID X 360 μm의 외경 모세관에 슬러리를 분배하며로서 현미경으로 관찰 모세 혈관이 달성된다 2-3mm 포장의 길이까지 입자로 채우고, 이것은 마이크로 반응 할 것이다 (그림 1) 20 μ.m의 ID 모세관은 키스톤 효과를 통해 마이크로 반응기에서의 입자를 유지한다. (15) </li><li> H 2 O / CH 3 CN 50:50 v / V를 50 μl의 솔루션 ~와 마이크로 반응기를 씻어하고 H 2 O / CH 3 CN (98)의 50 μl의 솔루션 ~와 : 2 v / V를. </li></ol> 샘플 솔루션의 2. 준비 주 : 유기 용매 및 산 및 용액 제제의 운반 작업 것을 포함 흄 후드에서 수행되어야한다. 고글, 장갑 및 보호 복을 착용 할 것. <ol><li> CH 3 OH 씻어 몇 유리 병 (4 ㎖) 및 폴리 프로필렌 튜브 (15 ml)에 건조. </li><li> H 2 O / CH 3 CN 용액 10 ㎖를 준비 (98 : 2 v / v)로 15 ml의 폴리 프로필렌 튜브에 200 μL의 CH 3 CN로 9.8 ml의에게 H 2 O를 혼합하여; 초음파로 섞는다. </li><li> 용액 10 ㎖를 준비 H 2 O / CH 3 CN (50:50 v / v)로 15 ml의 폴리 프로필렌 튜브에 5 ML의 H 2를 혼합하여 &lt;/ 서브&gt; O 5 ml의 CH 3 CN; 초음파로 섞는다. </li><li> H 2 O / CH 3 CN 4 ml의 4 μL TFA (10 %)를 추가하여 산성화 된 수용액 (TFA 0.01 %의 v / v)의 4 ml의 준비 : 4 ㎖의 유리 병에 (98 2 v / v)의; 초음파로 섞는다. </li><li> 4 ml의 H 2 O / CH 3 CN (50:50 v / v)로에서 4 mL 유리 바이알에 4 μl를 TFA (10 %)를 추가하여 산성화 된 유기 용액 (TFA 0.01 %의 v / v)의 4 ml의 준비; 초음파로 섞는다. </li><li> 16 mg의 NH 4 HCO 3 무게 4 mL 유리 바이알에서 분석 마이크로 밸런스와; (: 2 v / V를 98) 솔루션 및 초음파에 의해 분산 2 O / CH 3 CN에게 H의 4를 가하여 이 수성 버퍼 NH 4 HCO 3 (산도 ~ 7.8)의 50 mM의 솔루션을 초래한다. </li><li> 표준 단백질의 5 mg의 체중 (예를 들면, 소 혈청 알부민, 헤모글로빈 α / β, 시토크롬 C, 탄산 탈수 효소, α-2-HS 당 단백질 등) 4 mL 유리 바이알에서 분석 마이크로 밸런스와; 4 ML의 DI의 물 공급을 추가연구 및 초음파에 의해 분산; 이것은 일반적으로 높은 수준 μM 농도 용액을 초래한다. </li><li> NH 4 HCO 3 (50 mM)을 수성 완충액 적절한 부피 μM 높은 수준의 농도로 용액을 희석하여 1 mL의 단백질 용액 (1-2 μM)를 준비; 초음파로 섞는다. </li><li> (50 mM의 께) NH 4 HCO 3 170 μL 수성 완충액을 20 μg의 서열 분석 등급 트립신 용해 트립신 용액 (5 μM)를 준비; 초음파에 의해 분산. </li></ol> 3. 실험 설정 주 : LTQ-MS 시스템은 다양한 샘플 입력 방식으로 질량 분석 장치의 인터페이스 수있는 조립식 XYZ 스테이지를 포함하는 변성 ESI 공급원이 장착된다. <ol><li> 픽 조합에서 모세관 마이크로 반응기를 분리하고 PEEK 티에 연결합니다. </li><li> 픽 티 옆을 팔에 ~ 2cm 긴 백금 와이어를 삽입; 를 제공하는 1/32 &quot;PEEK 슬리브를 사용누수없는 연결과 백금 와이어를 절연합니다. </li><li> 픽 티의 반대쪽에 (~ 0.5 m 길이) 샘플 전송 모세관 50 μm의 ID X 360 μm의 외경을 연결합니다; 누출이없는 연결을 제공하기위한 1/32 &quot;PEEK 슬리브를 사용합니다. </li><li> XYZ 단계에 PEEK 티를 안전하고 멀리 질량 분석기 입구 모세관에서 ESI 터 ~ 2mm의 위치. </li><li> 질량 분광기의 ESI 전원부에 백금 와이어를 연결한다. </li><li> 스테인리스 연합 샘플 이송 모세관의 대향 단부에 연결; 누출이없는 연결을 제공하는 1/16 &quot;PEEK 슬리브를 사용한다. </li><li> 스테인레스 스틸 노동 조합의 반대 끝에 4-5cm 길이 1/16 &quot;PTFE 튜브를 연결하고 PTFE 튜브에 250 μL 주사기 바늘을 삽입, 예를 들어 (펌프를 켜고 원하는 값에 유량을 설정, 2 μL / 분). </li><li> 이동국 구을 제어하는​​ 소프트웨어 패키지에 입력 탠덤 MS 데이터 수집 매개분석을 수행하기위한 준비를 설치하는 방법 파일로 저장 거라고; LTQ-MS 시스템에 대해 다음 데이터 분석 의존 파라미터를 사용 제외 질량 폭 180 초 동안 활성화 된 동적 배제를 1.5 m / z ±; 하나의 MS는 한 줌 다음 스캔과 MS는 상위 5 가장 강렬한 봉우리 2 스캔, 5m / Z ± 스캔 폭을 확대; 충돌 유도 해리 분리 폭 3m / Z, 정규화 된 충돌 에너지 35 %, 활성화 Q 0.25 및 활성화 시간 30 밀리 초 설정 (CID) 매개 변수를 설정합니다. </li></ol> 4. 미세 유체 설치 <ol><li> 유리 또는 폴리머 기판으로부터 마이크로 유체 장치 준비, 입구에 접속 16-18 장치의 레이아웃이도 2a에 제공되고, 마이크로 반응기로 이루어진 (1) (0.13 mm 폭 × 2 mm 길이 × 50 μm의 깊이) (2 ), 배출구 (3) 측의 포트 (4); 유체 조작에 대한 상호 연결 채널은 ~입니다110 μm의 폭 ~ 50 μm의 깊이; 다중 채널 구조는 (5)로 이루어진 ~ 1.5 ㎛의 깊이 × 10 ㎛ 폭 X 위치 100 μm의 길이 25 ㎛의 미세 간격, 마이크로 리액터에 입자를 유지하기위한 필터로서 작용한다. </li><li> 또는 유체 전달 (그림 2B)에 대한 피팅을 수용하는 고분자 스탠드를 고안하여 입구 포트에 특수 커넥터를 접착하여 칩에 유체 전달 연결을 사용하도록 설정합니다. (16) </li><li> 단계 1.8에 기재된 바와 같이 입자 C18 (5 μm의)의 슬러리를 조제; 칩 측 포트에 접속 1/16 &quot;PTFE 튜빙을 사용하여 주사기와 마이크로 반응기에 상기 슬러리를 제공하는 것은 (4) 90 ºC 오븐에서 에폭시 접착제와 1 시간 동안 경화와 함께 입자 로딩 후 포트를 밀봉 (16). </li><li> 경화 O / N을 E6000 접착제로 밀봉하고,하자, 출구에 모세관 (20 μm의 ID X 90 μm의 OD × 10 mm 길이)를 삽입; 이합니다 (ESI의 터가 될 것이다 <strong&gt; 6). </li><li> 50㎛의 ID X 360 μm의 지름을 연결 (~ 0.5 m 길이) 칩 유입구 샘플 이송 모세관 (2); 3.6-3.7에 기재된 바와 같이, 250 μL 주사기 및 주사기 펌프 모세관의 대향 단부를 연결한다. </li><li> 흡기구 직전 샘플 전송 라인상의 경량 PEEK 티 커넥터 배치 (2) 및 티 측 아암 편에 와이어를 삽입 3.2에 기재된 바와 같고; 이것은 ESI 전극 될 것입니다. </li><li> 질량 분석기 입구 모세관에서 ~ 2mm 거리에 위치하는 ESI 이미 터와 XYZ 스테이지에 미세 유체 소자를 고정; 3.8에 기재된 바와 같이 분석을 위해 MS를 준비한다. </li></ol> 5. 샘플로드, MS 분석을위한 단백질 분해 소화 및 용출 모든 용액 / 샘플 전송 단계를 주사기 펌프의 도움으로 수행되고, 데드 있습니다 volum 보상하기 위해, 완료하기 위해 몇 분 동안 추가로 허용한다 참고및 상기 마이크로 반응기에서 이송 라인과 관련된 ES; 필요한 시간이 포함 된 유량에 따라 달라집니다. <ol><li> 마이크로 반응을 씻어 2 O / CH 3 CN H의 수성 NH 4의 솔루션 HCO 3 (50 mm)를 주입하여 분석 준비를 5 분 동안 2 μL에서 (98 2 v / v)의 / 분; 250 μL 주사기를 사용합니다. </li><li> 2 μL의 시료 용액을 주입하여 미세 반응기에 단백질 샘플 (1 μM)을로드 / 분, 5 분; 250 μL 주사기를 사용합니다. </li><li> 1-3 분 / 2 μL에서 분 마이크로 리액터에 흡착 된 단백질을 통해 트립신 용액 (5 μM)을 주입. </li><li> TFA의 산성화 수용액으로 마이크로 반응기를 세척하여 단백질 분해 소화 과정을 끄다 (0.01 % v / v)의 H 2 O / CH 3 CN에서 (98 : 2 v / v)의 2 μL / 분 5 분; 250 μL 주사기를 사용합니다. </li><li> (단백질의 TFA 산성화 된 유기 용액을 주입하여 미세 반응기로부터 0.01 % 다이제스트 용출 시작v / v)로 H 2 O / CH 3 CN에서 (50:50 v / v)의 300 NL / 분. </li><li> 단말기 데이터 수집 과정과 ESI 전압 (~ 2000 V)를 켭니다; 단계 3.8에 제공된 데이터 수집 매개 변수를 사용하여 MS 데이터를 획득; 트립신 펩타이드의 용출을 관찰합니다. </li></ol> 6. 데이터 처리 <ol><li> 원시 MS 데이터 포맷과 호환되는 검색 엔진 탠덤 MS 데이터를 처리한다. <ol><li> 생체의 특정 최소 중복 단백질 데이타베이스를 사용하여 LTQ-MS 원시 파일을 처리; 검색 엔진의 원시 데이터를 업로드, 각각 2 및 1 다에 부모 및 조각 이온 질량 공차를 설정하고, 최대 두 개의 검색에 대한 분열을 놓친 만 완전히 트립신 단편을 사용한다; 번역 후 변형을 허용하지 않습니다 각각 1 %와 3 %로 높은 및 중간 신뢰 거짓 발견 비율 (FDR)를 설정하고, 번역 후 변형을 허용하지 않습니다. </li></ol></li><li> 만 높은 신뢰를 선택하기 위해 데이터를 필터링펩타이드는 데이터베이스의 단백질과 일치; 단백질 및 펩타이드 수준의 결과를 평가합니다. </li></ol>

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The authors declare no competing financial interests.

이 작품에서 설명하는 마이크로 반응기가 제공하는 간편한 구현 미만 30 분에서 MS 분석 및 식별을 가능하게하는 단백질의 효소 소화를 수행하기위한 실험 장치를. 기존의 방법에 비해이 시스템의 뚜렷한 장점은 단순성, 속도, 낮은 시약 소모와 낮은 비용을 포함한다. 특히, 고가의 트립신 고정화 비드 카트리지가 필요 없다. 모세관 마이크로 리액터의 제조는 간단 (도 1a), 그리고 통상...

정제 된 단백질의 동정 및 특성화 자주 MS 기술을 사용함으로써 달성된다. 단백질은 효소로 분해되고 그 펩티드는 더 간단한 주입 실험 장치를 사용하여 MS로 분석된다. 단백질 분해 소화 가장 MS 분석기의 유효 질량 범위에 속하는 작은 펩티드 단편을 생성하기 위해 필요하고, 그 용이 아미노산 서열 정보를 생성하기 위해 저 에너지 충돌 유도 된 분해를 통해 단편화 될 수있다. 절연 단백질 또는 단순 단백질 혼합물, MS 검출하기 전에 펩티드의 크로마토 그래피 분리에 대한 추가적인 필요가 없다. 25-50 펩티드의 혼합물을 용이 MS 이온 소스에 직접 주사기 펌프 시료를 주입하여 분석 할 수있다. 질량 분석 장치는 분석을 수행하고, 짧은 시간 프레임 내에서 단백질의 서열을 확인할 수있다. 현대 데이터 수집 방법과,이 프로세스는 승 달성 될 수있다몇 분 또는 초 ithin. 단시간 스케일에 대한 전체 프로세스를 완료하는 제한 요인이 단백질 분해성 분해 공정이다. 일반적으로, 이것은 수 시간에 걸쳐 수행된다 (또는 O / N) 용액에 37 ºC에서 사용한 기판 (50-100) 효소의 비를 1. 설명 된 미세 반응기 또는 시판되는 카트리지의 형태로, 분, 초, 고정화 효소의 마이크로 리액터의 효소 소화 시간을 감소시킨다. 1-6 일반적으로 효소를 공유 결합, 비 - 공유 / 물리 흡착 복합체가 고정화 형성 또는 캡슐화, 효소 처리의 -3,6- 향상된 효율으로 사용되는 큰 표면 대 부피 및 효소에 대한 기판 비. 고정화 반응기의 추가 이점은 MS 분석에서자가 분해 효소와의 간섭을 감소 효소 안정성 및 재사용 성을 향상시킬 것을 포함한다. 접근법을 이용하여 유리 또는 중합 미세 다양한 장치가 설명되었지만항체 - 항원 상호 작용에 의해 자성 비드에 고정화 효소를 이용하여, 7,8- 9는 티타니아, 알루미나 졸 - 겔 10 nanozeolites 11에 캡슐화 또는 니켈 NTA 또는 그의 태그 복합체 형성을 통해 캡처 된 금 나노 입자의 네트워크에 포획. 6 대안 고정화 효소 개방 관형 모세관도 개발되어있다. (12) 또한, 향상된 단백질 분해 절단은 30-120에 대한 반응 시간을 감소시키기위한 제어 마이크로파 조사 (13) 또는 압력 보조 또는 압력 사이클링 기술 (PCT)을 사용하여 입증되었다 분. (14) 고정화 효소 반응기의 여러 장점에도 불구하고, 상업적 카트리지의 비용이 높고, 일상적인 사용을위한 미세 유체 장치의 유용성은 제한되어, 추가 계측을 필요로하는 전자 또는 PCT 기술 결과의 용도. 이 연구의 목적은 circumve 방법을 개발했다이러한 단점을 NTS, 그것은 쉽게 분 이내 MS 분석에 대비 단백질의 효소 적 분해를 수행하기위한 간단하고 효과적인 방법으로 연구자 강화할 각 실험실에서 구현 될 수있다. 접근법은 모세관 또는 미세 유동 장치에 사전로드 된 소수성 C18 입자의 사용에 의존하며 통해 효소의 주입 중에 효소 분해 한 다음 이들 입자에 관심 단백질 (들)의 흡착 충전 층 및 캡처 단백질 (들). 이 방법에서, 기판은 비 - 공유 상호 작용을 통해 고정되고, 상기 효소가 고정화 된 단백질을 통해 주입된다. 단백질 분해 소화 효율을 효소 처리, 환원 및 입자의 표면으로부터의 거리와, 확산 시간은 단백질을 노출 큰 입자 표면적이 증가 질량 전사 효소의 활성에 영향을 미칠 수없는 공유 결합, 기능 향상 빠르게 evaluat다른 효소 처리 성 및 다중화 E의 조합이 과정은 미세 형식으로 실행되는 경우. 이 방법은 표준 단백질 및 ESI-MS 검출 이전에 단백질 분해 소화 트립신 가장 일반적으로 사용되는 효소의 혼합물을 이용하여 설명된다. 본 연구에 사용 된 검출 질량 분석기는 선형 트랩 중극 (LTQ)기구이다.

<table>
	<tbody>
		<tr>
			<td>Ion trap ESI-MS</td>
			<td>Thermo Electron</td>
			<td>LTQ</td>
			<td>The LTQ mass spectrometer is used for acquiring tandem MS data</td>
		</tr>
		<tr>
			<td>XYZ stage</td>
			<td>Newport</td>
			<td>Multiple parts</td>
			<td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Edmund optics</td>
			<td>G81-278</td>
			<td>The microscope is used to observe the microreactor packing process</td>
		</tr>
		<tr>
			<td>Analytical balance/Metler</td>
			<td>VWR</td>
			<td>46600-204</td>
			<td>The balance is used to weigh the protein samples</td>
		</tr>
		<tr>
			<td>Ultrasonic bath/Branson</td>
			<td>VWR</td>
			<td>33995-540</td>
			<td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td>
		</tr>
		<tr>
			<td>Syringe pump 22</td>
			<td>Harvard Apparatus</td>
			<td>552222</td>
			<td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td>
		</tr>
		<tr>
			<td>Milli-Q ultrapure water system&#160;</td>
			<td>EMD Millipore</td>
			<td>ZD5311595&#160;</td>
			<td>The MilliQ water system is used to prepare purified DI water</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (1000 &#181;L)</td>
			<td>VWR</td>
			<td>53513-410</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (100 &#181;L)</td>
			<td>VWR</td>
			<td>53513-406</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (10 &#181;L)</td>
			<td>VWR</td>
			<td>53513-402</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Fused silica capillary (100 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP100375</td>
			<td>This capillary is used for the fabrication of the microreactor</td>
		</tr>
		<tr>
			<td>Fused silica capillary (20 &#181;m ID x 100 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP020090</td>
			<td>This capillary is used for the fabrication of the ESI emitter</td>
		</tr>
		<tr>
			<td>Fused silica capillary (50 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP050375</td>
			<td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td>
		</tr>
		<tr>
			<td>Glass capillary cleaver</td>
			<td>Supelco</td>
			<td>23740-U</td>
			<td>This is a tool for cutting fused silica capillaries at the desired length</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Eclectic Products</td>
			<td>E6000 Craft</td>
			<td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Epo-Tek</td>
			<td>353NDT</td>
			<td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td>
		</tr>
		<tr>
			<td>Reversed phase C18 particles (5 &#181;m)</td>
			<td>Agilent Technologies</td>
			<td>Zorbax 300SB-C18</td>
			<td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4 mm x 20 cm C18 LC column from Agilent</td>
		</tr>
		<tr>
			<td>Syringe/glass (250 &#181;L)</td>
			<td>Hamilton</td>
			<td>81130-1725RN</td>
			<td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td>
		</tr>
		<tr>
			<td>Internal reducing PEEK Union (1/16&#8221; to 1/32&#8221;)</td>
			<td>Valco</td>
			<td>ZRU1.5FPK</td>
			<td>This union is used to connect the 250 &#181;L syringe to the microreactor for loading the 5 &#181;m particle slurry</td>
		</tr>
		<tr>
			<td>Stainless steel union (1/16&#8221;)</td>
			<td>Valco</td>
			<td>ZU1XC</td>
			<td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td>
		</tr>
		<tr>
			<td>Microvolume PEEK Tee connector (1/32&#8221;)</td>
			<td>Valco</td>
			<td>MT.5XCPK</td>
			<td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td>
		</tr>
		<tr>
			<td>Tee connector (light weight)</td>
			<td>Valco</td>
			<td>C-NTXFPK</td>
			<td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td>
		</tr>
		<tr>
			<td>Pt wire (0.404 mm)</td>
			<td>VWR</td>
			<td>66260-126</td>
			<td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td>
		</tr>
		<tr>
			<td>PTFE tubing (1/16&#8221; OD)</td>
			<td>Valco</td>
			<td>TTF115-10FT</td>
			<td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&#160;</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8220; ID x 1/16&#8221; OD)</td>
			<td>Upchurch Scientific</td>
			<td>1565</td>
			<td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50 &#181;m ID transfer capillary</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8221; ID x 1/32&#8221; OD)</td>
			<td>Valco</td>
			<td>TPK.515-25</td>
			<td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td>
		</tr>
		<tr>
			<td>Clean-cut polymer tubing cutter</td>
			<td>Valco</td>
			<td>JR-797</td>
			<td>This cutter is used to pre-cut the 1/16&#8221; and 1/32&#8217; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td>
		</tr>
		<tr>
			<td>Amber vial (2 mL)</td>
			<td>Agilent&#160;</td>
			<td>HP-5183-2069</td>
			<td>The vials are used to prepare sample solutions and the C18 particle slurry&#160;</td>
		</tr>
		<tr>
			<td>Amber vial (4 mL)</td>
			<td>VWR</td>
			<td>66011-948</td>
			<td>The vials are used to prepare sample solutions</td>
		</tr>
		<tr>
			<td>Polypropylene tube (15 mL)</td>
			<td>Fisher</td>
			<td>12-565-286D</td>
			<td>The vials are used to prepare buffer solutions</td>
		</tr>
		<tr>
			<td>Cylinder (100 mL)</td>
			<td>VWR</td>
			<td>24710-463</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Cylinder (10 mL)</td>
			<td>VWR</td>
			<td>24710-441</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>83007-386</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (100 &#181;L)</td>
			<td>VWR</td>
			<td>53503-781</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (10 &#181;L)</td>
			<td>VWR</td>
			<td>53511-681</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Glass substrates</td>
			<td>Nanofilm</td>
			<td>B270 white crown, 3&#8221; x 3&#8221;</td>
			<td>These are glass substrates for microchip fabrication</td>
		</tr>
		<tr>
			<td>Male nut fitting (1/16&#8221;)</td>
			<td>Upchurch</td>
			<td>P203X</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td>Nanoport assembly</td>
			<td>Upchurch</td>
			<td>N-122H</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein standards</td>
			<td>Sigma</td>
			<td>Multiple #</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC grade</td>
			<td>Fisher</td>
			<td>A955</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC grade</td>
			<td>Fisher</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, HPLC grade</td>
			<td>Sigma</td>
			<td>650447</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Aldrich</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

질량 분석 (MS)의 대부분은 단백질 분석 방법은 일반적으로 트립신, 검출 효소 분해 단계 전에 수반 기반. 이 단계는 질량 분석 기기의 유효 주사 범위 내에 MW &lt;3,000-4,000 다 함께 일반적으로 분자량이 작은 펩타이드의 생성을 위해 필요하다. 종래의 프로토콜은 37 ºC에서 O / N 효소 소화를 포함한다. 최근 발전은 전형적으로 예를 들면, 전자 렌지 나 고 고정화 효소 또는 몇 분에 단백질 가수 분해에 필요한 시간을 단축 상보 물리적 처리 (범위의 마이크로 리액터의 이용을 포함하는 다양한 전략의 개발을 주도 압력). 본 연구에서는 단백질을 빠르게 분해 효소를 달성하기 위해 임의의 실험실에서 구현 될 수있는 간단하고 비용 효율적인 방법을 설명한다. 단백질 (또는 단백질의 혼합물)은 결합 된 C18 역상 높은 퍼포에 흡착모세관 컬럼에서 사전로드 된 액체 크로마토 그래피 (HPLC) 실리카 입자를 필요치 않는, 수성 완충액 트립신은 짧은 시간 동안 입자를 통해 주입된다. 온라인 MS 검출을 사용하려면 트립신 펩티드는 MS 이온 소스에 직접 증가 유기물 함량과 용매 시스템과 용출된다. 이 방법은 고가의 고정화 효소 입자의 사용을 회피하고, 처리를 완료하는 처치를 필요로하지 않는다. 단백질 소화 완전한 샘플 분석은 각각 ~ 30 분 이하 ~ 3 분 이상으로 달성 될 수있다.

단백질 분해 소화 공정의 대표적인 결과가 상기 마이크로 반응기 (도 1, 2) 표 1에 제공하여, 단백질의 혼합물을 동시에 수행 하였다.이 표는 특정 단백질을 식별하는 고유의 펩티드 서열을 포함하는 십자가 상관도 스코어 (Xcorr) (즉, 대응하는 탠덤 질량 스펙트럼 실험 투 이론적 매치의 품질 특징 스코어) 놓친 분열 횟수, 펩티드 충전 상태 (z...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

플로우를 통해 마이크로 반응기와 MS 검출을위한 단백질의 빠른 효소 처리

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K 조회수.  Virginia Tech. A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

비디오: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

연구

생체공학

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

낮은 풍부 단백질을 검출하기위한 플라즈마 또는 소변의 하이드로 겔 나노 입자 수확

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

미세 유체 정밀 작은 볼륨 샘플 처리를위한 플랫폼 및 음향 마이크로 디바이스로 구분 생물 입자 크기의 사용

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRET 유동 세포 계측법과 Proteopathic의 시드 활동의 민감한 검출

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

동맥 경화의 빠른 검색을위한 산화철 나노 입자의 전자 레인지 중심의 합성

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

세포의 초고속 자화에 대한 양이온 Magnetoferritin의 합성

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

플로우를 통해 마이크로 반응기와 MS 검출을위한 단백질의 빠른 효소 처리

요약

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