This research was generously funded by the NIH NCI IMAT Program Grant R33 (CA147988-02), the NIH NIGMS Graduate Training in Molecular Biotechnology at NC State Grant (T32GM008776), the US Dept. of Education GAANN Fellowship Program in Molecular Biotechnology at NC State Grant (P200A140020), the W.M. Keck Foundation, and North Carolina State University. Hen plasma was obtained with the assistance of Dr. James N. Petitte and Rebecca Wysocky in the NC State University Dept. of Poultry Science.

<ol>
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We have a collaborative relationship with Cambridge Isotope Laboratories (CIL) to disseminate the P2GPN reagent and technology to the broader scientific community in the form of a product (GTK-1000). However, we do not receive any revenue based on the sale of this product.

High-throughput quantitative methods are needed to facilitate routine glycan analysis. For the last thirty years, glycomics analysis has been limited to a subset of research groups, despite its importance in disease, clinical applications, and pharmaceuticals. The FANGS-P2GPN purification and tagging method for glycomics and proteomics performs the same analysis on a single aliquot of sample, reducing the cost of supplies and the amount of material needed (particularly important in human and mouse studies). Furthermore, efforts to minimize variability in preparations are critically important, as every additional step contributes to error, potentially masking important but low-abundant changes in case-control studies. Coupling of FANGS to hydrophobic hydrazide tagging allows protein and glycan samples to be run on the same RPLC column, enhances glycan ionization, provides for relative quantification, and can be quantitatively applied to plasma.
For N-glycan analysis, it is critical to use the suggested level of PNGase F to achieve full de-glycosylation. Though glycans are solvent exposed, denaturation of proteins and excess enzyme help ensure efficient and complete cleavage. For accurate quantitation of the glycans, it is necessary to ensure that they are completely dried after derivatization to quench the reaction and prevent cross-reactions when mixing the NAT and SIL species. Finally, when extending the workflow to glycosite analysis, timing of the steps is critical to minimize non-specific deamidation. The modified protocols provided for combined glycomics and proteomics analysis work consistently when performed accordingly.
The workflow achieves accurate relative quantitation of N-glycans from plasma compared to the gold-standard, SPE method. There is no apparent bias in the types of glycans extracted in terms of molecular weight, hydrophilicity, and compositional structure. Though we have not explored the qualitative analysis of O-linked glycans, we expect that FANGS could accommodate the addition of a &#946;-elimination step post-PNGase F digestion of N-linked glycans. However, procedures would require significant modification for reagent cleanup prior to mass spectrometry, and peptide analysis will be significantly impacted. For proteomics, the same depth of proteome coverage is achieved compared to traditional FASP methods. Importantly, methods achieve a minimal false discovery rate for N-glycan deamidation. While the method is compatible with 18O labeling of Asn during the PNGase digestion step22,23, the low glycosylation site false discovery rate suggests that it may not be necessary, further reducing costs and complexity.
The proteome is not enriched for glycoproteins in this method, which has both advantages and disadvantages. Certain low abundant glycoproteins may not be detected in the analysis. However, the occupancy of glycosylated sites per protein, can be compared between biological samples. Additionally, the error and bias introduced from lectin affinity purification or chemical enrichment is eliminated. In conclusion, coupling of FANGS to the individuality normalization when labeling with glycan hydrazide tags strategy results in a simplified, quantitative, high-throughput method for the tandem analysis of the glycome and proteome with great potential for application in clinical case-control studies.

프로테오믹스 분야에서는, 필터를 이용한 시료 준비 (FASP)가 널리 출발 물질의 양을 최소화하는 샘플 준비 아티팩트를 감소시키고, 시료 처리 1을 최대화하는 기능을 채용하고있다. 그러나, 이러한 방법은 아직 나오고 글라 이코믹스의 필드 견인력을 얻을 수있다. 높은 처리량의 개발은 양적 워크 플로우 때문에 필수적인 생물학적 방어 당화의 역할과 암 또는 질병 2,3에 의해 그 변조의 필요하다. 포유 동물에서, N의 -glycans은 코어 구조를 장식 당 단위 (탄당 (육각), hexosamines (HexNac), 시알 산 (NeuAc의), 그리고 푸 코스 (FUC)), (육각 3 HexNac 2) 4 공유 결합을 반복으로 구성되어 아스파라긴합니다. 이성질체가 반영되면 glycospace 상당히 큰 것이지만 (&gt; 10 ~ 12)은, 그 조성에 기초하여 매우 작고, 전형적으로는 분자량 1,000-8,000 다 5 &lt;에서부터/ SUP&gt;. 클래스의 조성 균일 성 및 글리 칸의 친수성 정제 분리에 고유 한 문제를 제기하고, 질량 분석 (MS) (6)를 워크 플로우. 전통적으로, N의 -glycans는 F (MALDI 스펙트럼은 PNGase F)는 펩티드 - N으로 단백질 또는 펩티드의 글리코시다 제 분해 된 후 히드라 지드 비드 (8)에 의해, 렉틴 친 화성 크로마토 그래피 (7)에 의해 농후 포착, 또는 고체 상 추출 (SPE) 9,10 통해 정제. 이들 방법은 모두 매우 효과적이지만, 이들은 탈염위한 추가 단계가 도입 된 샘플의 수는 동시에 처리 제한한다. 지난 10 년간, 글라 이코믹스에 대한 높은 처리량 플랫폼 다수 제안되어있다. 김 외. 진공 조작, SPE 96 웰 플레이트 (11)를 사용하여 반 - 자동화 된 방법을 발표 하였다. 또한, 친 화성 필터 방법 (N의 -glyco-FASP)는 파이의 초기 유도체를 필요로하는 맨 그룹에 의해 개발되었다렉틴 (12)의 합성과 LTER. 마지막으로, 토마스 - 오우 츠 그룹은 glycospace (13)의 좁은 조성 크기를 악용 반 정량적 방법, N -Glycan 분리 (송곳니)를 돕는 필터를 제안 하였다. 분자량 컷 - 오프 필터에 의존 작은 오염물 제 낭비 세척 한 다음 N의 -glycans는 분해 용출 하였다. 탈글 라이코 실화 된 단백질이 프로토콜에서 필터에 남아 인라인 FASP을 실시 할 수있다. 식별 및 전기 분무 이온화에 의해 글리 칸의 정량화 (ESI) MS는 저 풍부한 종의 검출을위한 이성체 및 유도체의 (부분) 해결을위한 오프라인 분리가 필요합니다. 개성 라벨링 당쇄 히드라 지드 태그 전략 정규화 역상 액체 크로마토 그래피 (역상) (14, 15)과의 호환성을 부여한다. 4- 펜 에틸 benzohydrazide (P2PGN)은 소수성 태그 ENH, 글리 칸의 친수성을 매개평균적으로 이온화 ancing 16 4 배. 용이 한 조건에서 화학량 론적 1 ​​: 메틸화 예 17 아민 반응성 화학 태그 (18)과 같은 다른 기술은 유사한 이점을 제공하지만, 히드라 지드의 반응에 글리 칸은 (1) 반응한다. 상대 정량 네이티브 (NAT) 또는 (13) C (6) 안정된 동위 원소 라벨 (SIL)로 유도 샘플의 직렬 분석에 의해 달성된다. 다음 방법은 정확한 상대 정량 P2GPN 소수성 태그에 플라즈마 응용 프로그램과 커플을위한 송곳니를 진화. 또한,이 분석의 무결성을 손상시키지 않고, 샘플의 단일 나누어지는에 총 총의 프로테오믹스, 탈 아미드 화 프로파일, 양적 글라 이코믹스을 수행하도록 설계되었습니다.

<table><tbody><tr><td>Acetic Acid (50%)</td><td>Sigma Aldrich&amp;nbsp;</td><td>45754</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH015-4</td><td></td></tr><tr><td>Ammonium Bicarbonate</td><td>Sigma Aldrich&amp;nbsp;</td><td>A6141</td><td></td></tr><tr><td>Bradford Reagent</td><td>Sigma Aldrich&amp;nbsp;</td><td>B6916</td><td>Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1)</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich&amp;nbsp;</td><td>C1016</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 R</td><td>Alternate centrifuges that reach 14,000 x g are suitable</td></tr><tr><td>DL-Dithiothreitol, 1&amp;nbsp;M in solution</td><td>Sigma Aldrich&amp;nbsp;</td><td>646563</td><td></td></tr><tr><td>Easy-nLC 1000</td><td>Thermo Scientific</td><td>LC120</td><td>Alternate nano or ultra high pressure LCs will produce similar data, such as: 1. Dionex UltiMate&amp;Ograve; 3000 LC (Thermo Scientific); 2. Acquity UPLC (Waters).</td></tr><tr><td>Floating Tube Rack</td><td>TedPella</td><td>20831-20</td><td></td></tr><tr><td>Fetuin</td><td>New England Biolabs&amp;nbsp;</td><td>P6042S</td><td></td></tr><tr><td>Fisher Scientific Isotemp Standard Lab Ovens</td><td>Fisher Scientific</td><td>11-690-625F</td><td>Alternate incubators that reach 56&amp;nbsp;&amp;deg;C are suitable</td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich&amp;nbsp;</td><td>56302</td><td></td></tr><tr><td>GE Microwave Oven</td><td>General Electric</td><td>57B5 E82904</td><td>Any microwave with adjustable power settings is suitable</td></tr><tr><td>INLIGHT Glycan Tagging Kit&amp;nbsp;</td><td>Cambridge Isotope Laboratories</td><td>GTK-1000</td><td>The INLIGHT kit provides NAT and SIL versions of the P2GPN reagent.</td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich&amp;nbsp;</td><td>A3221</td><td></td></tr><tr><td>Kinetix 2.6 mM, 100 &amp;Aring;, C&lt;sub&gt;18&lt;/sub&gt; bulk stationary phase</td><td>Phenomenex&amp;nbsp;</td><td>Bulk Media</td><td>Alternative: Any C&lt;sub&gt;18&lt;/sub&gt; stationary phase 5&amp;nbsp;mM</td></tr><tr><td>Mascot Daemon Software and Server</td><td>Matrix Science</td><td></td><td>Alternative: Proteome Discoverer Software (Thermo Scientific)</td></tr><tr><td>Methanol, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH230-4</td><td></td></tr><tr><td>PicoFrit Self-Pack Column: 360&amp;nbsp;&amp;#956;m, OD 75&amp;nbsp;&amp;#956;m ID, 15&amp;nbsp;&amp;#956;m tip, non-coated, 5 per box, 50 cm</td><td>New Objective</td><td>1 5 PF360-75-15-N-5</td><td></td></tr><tr><td>PNGase F (glycerol-free), 75,000&amp;nbsp;units/ml</td><td>New England BioLabs</td><td>P0705L</td><td></td></tr><tr><td>Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Scientific</td><td></td><td>Alternate high mass accuracy (5&amp;nbsp;ppm) mass spectrometers will provide similar data</td></tr><tr><td>RNase B</td><td>New England Biolabs&amp;nbsp;</td><td>P7817S</td><td></td></tr><tr><td>Trypsin from Porcine Pancreas</td><td>Sigma Aldrich&amp;nbsp;</td><td>T6567-5X</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich&amp;nbsp;</td><td>51456</td><td></td></tr><tr><td>Vacuum ConcentratorSavant SPD131DDA SpeedVac Concentrator</td><td>Thermo Scientific</td><td>SPD131DDA</td><td>Alternate vacuum concentrators are suitable</td></tr><tr><td>Vivacon 500 30 kDa Filters&amp;nbsp;</td><td>Sartorius Stedim Biotech</td><td>VN01H22</td><td>Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10&amp;nbsp;kDa Membrane (Millipore UFC501096)</td></tr><tr><td>Water, HPLC grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH365-4</td><td></td></tr><tr><td>Water, &lt;sup&gt;18&lt;/sup&gt;O</td><td>Cambridge Isotope Laboratories</td><td>OLM-240-97-1</td><td>The addition of &lt;sup&gt;18&lt;/sup&gt;O in the PNGase F digest step is optional and may not be necessary for deamidation studies completed with 95% confidence</td></tr><tr><td>Xcalibur 2.0</td><td>Thermo Scientific</td><td>XCALIBUR20</td><td></td></tr><tr><td>Zwittergent Test Kit</td><td>Merck Millipore</td><td>693030</td><td></td></tr></tbody></table>

a quantitative glycomics and proteomics combined purification strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

<img alt="그림 1" src="/files/ftp_upload/53735/53735fig1.jpg" /> 그림 1 :. 결합 단백질 체학 및 글라 이코믹스 분석을위한 송곳니 - P2GPN 소수성 태그 결합 방법의 체계 (방법)이 주어 당화 사이트 식별과 글라 이코믹스 전용 처리 및 직렬 글라 이코믹스 및 프로테오믹스 분석,이 서로 다른 단계, 강조 표시됩니다. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 이 인라인 프로테오믹스 및 글라 이코믹스 필터 기반 P2GPN 소수성 태깅 방법 (방법 A)는 풀링 암탉 혈장 샘플 (도 1)를 이용하여 식별, 정량 및 분자량 바이어스 검증 하였다. 전적으로 글라 이코믹스 실험의 경우, N의 존재비에 간 비교는 방법에 의해 추출 -glycans이 SPE (금 표준, 방법 B) 만들어졌다 (그림 2) carbograph합니다. 두 가지 방법 사이의 글리 칸의 존재비에서 유의 한 차이가 없었다. 동일한 방법에 의해 추출 된 글리 칸의 NAT 비율 내 변동은 SIL을 정량함으로써 측정 하였다. 두 방법의 로그 2 -distributions 제로를 중심으로, 가우스했고, (그림 3A-B) 유의 한 차이가. 분자량은 필터가 N이 분자량 범위 및 친수성 (도 4)에 기초 -glycans 구별하지 않았다 시사 완전히 겹쳐진 두 프로토콜 사이의 범위이다. <img alt="그림 2" src="/files/ftp_upload/53735/53735fig2.jpg" /> 그림 2 :. NAT 및 SIL N의 몰 혼합물 방법 또는 방법 B에 의해 추출 -glycans 2.5 암탉 플라즈마의 μL (N = 4) 샘플이었다 항문 제조 하였다각 NAT / SIL의 채널 부 (6) 각각의 당쇄에 대한 존재비 제안 추천 파라미터에 따른 UPLC-MS에 의해 yzed 분자를 보정 추출 된 이온 크로마토 그램 (MMA = 3 PPM) 아래의 면적을 적분하여 계산 하였다 TGNF 중량 중복 및 조정. 이 비율은 표준 오류가 표시됩니다. 방법 B의 존재비 :. 방법 N의 -glycans은 (p&gt; 0.05) 유의 한 차이가 없었다 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53735/53735fig3.jpg" /> 도 3 (A) 방법 인트라 변이 (N = 133) 또는 (B) 방법은 B (N = 123) 전략을 비교 하였다. 같은 추출 방식에서 N의 -glycans의 NAT 및 SIL 몰 혼합물세 가지 기술 복제 통해 분석 하였다. 로그 2 -distributions 제로를 중심으로 두 흐름 사이. 유의 한 차이가 없었다 된 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53735/53735fig4.jpg" /> 도 4 각 전략 글리 칸에서의 분자량 범위를 비교 하였다. 이 워크 플로는 동일한 분자량 범위에 걸쳐 글리 칸을 얻었다. N은 다른 단지 검출 (1 × 10 5 풍부)의 한도를 초과 하락에 비해 하나의 프로토콜에서 검출 -glycans하고 체계적인 편견을 반영하지 않습니다. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">더 큰 보려면 여기를 클릭하십시오 이 그림의 버전입니다.</a> (표 5)가없는 전통 FASP 제제를 비교 하였다. FASP 트립신 다이제스트 (표준 프로토콜) 다음에 우리의 일반적인 18 시간 필터를 기반으로 MALDI 스펙트럼은 PNGase F 소화는 glycosites의 식별을 방해 할 수 비특이적 탈 아미드 화 상당한 수준의 결과. 따라서, 높은 온도 (50 °의 C)와 MALDI 스펙트럼은 PNGase F 스파이크 - 단계 (SPI 프로토콜)에서 짧은 배양 시간을 이용하는 방법은 마이크로파 분해 프로토콜 (MD 프로토콜)와 함께 대안으로 탐구하고, 비 최소화 특정 탈 아미드 화. 새로운 소화 방법에서 관찰 글리 칸은 표준 18 시간에서 조성물 또는 존재비에서 유의 한 차이가 없었다 37 ° C 필터 MALDI 스펙트럼은 PNGase F 다이제스트 ( <strong&gt; 그림 5). 짧은 트립신 배양과 결합, 비특이적 탈 아미드 화 크게 glycosites &lt;5 % (표 5)에 대한 거짓 긍정적 인 속도로 감소 하였다. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td><td colspan="4"> 단백질 </td><td></td><td colspan="4"> 펩티드 </td><td></td><td colspan="4"> 탈 아미드 펩티드 </td><td></td><td colspan="4"> Glycosites </td><td></td><td colspan="4"> 당 단백질 </td></tr><tr><td colspan="2"></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td></tr><tr><td colspan="2"> FASP </td><td colspan="2"></td><td colspan=&quot;2&quot;&gt; (249) </td><td></td><td colspan="2"></td><td colspan="2"> 3083 </td><td></td><td colspan="2"></td><td colspan="2"> 795 </td><td></td><td colspan="2"></td><td colspan="2"> (5) </td><td></td><td colspan="2"></td><td colspan="2"> (5) </td></tr><tr><td colspan="2"> 18hr </td><td colspan="2"> (217) </td><td colspan="2"> (304) </td><td></td><td colspan="2"> 6013 </td><td colspan="2"> 5086 </td><td></td><td colspan="2"> 1,029 </td><td colspan="2"> 733 </td><td></td><td colspan="2"> 257 </td><td colspan="2"> (24) </td><td></td><td colspan="2"> (112) </td><td colspan="2"> (18) </td></tr><tr><td colspan="2"> MD </td><td colspan="2"> (270) </td><td colspan="2"> (266) </td><td></td><td colspan="2"> 5190 </td><td colspan="2"> 5125 </td><td></td><td colspan="2"> 465 </td><td colspan="2"> (455) </td><td></td><td colspan="2"> (254) </td><td colspan="2"> 8 </td><td></td><td colspan="2"> (102) </td><td colspan="2"> (7) </td></tr><tr><td colspan="2"> SPI </td><td colspan="2"> 281 </td><td colspan="2"> 288 </td><td></td><td colspan="2"> 4729 </td><td colspan="2"> 4,482 </td><td></td><td colspan="2"> (602) </td><td colspan="2"> 573 </td><td></td><td colspan="2"> (232) </td><td colspan="2"> (10) </td><td></td><td colspan="2"> (145) </td><td colspan="2"> 8 </td></tr></tbody></table> . 표 5 : 표준 트립신에서 단백체 데이터의 비교는 인라인 송곳-P2GPN 태깅 방법 만들었다 대 소화 물질을 사용하여 수행 하였다 (+) 및하지 (-) MALDI 스펙트럼은 PNGase F는 비특이적 탈 아미드 화 및 추정의 배경 요금을 결정 거짓 긍정적 인 요금 glycosite. 단백질을 1 % 거짓 검색 속도 (FDR)로 확인되었다; 펩타이드는 프로테옴 Discoverer는 1.4 &quot;높은&quot;펩타이드의 신뢰를 기반으로 여과 (Q &lt;0.01); glycosites는 identifi했다에드 보존 글리코 실화를 모티브로 (NXS / T)의 탈 아미드 화를 포함하는 고유 한 시퀀스에 기초 및 당 단백질은 적어도 하나의 식별 glycosite 확인 된 단백질을 함유하는 것으로 정의 하였다. <img alt="그림 5" src="/files/ftp_upload/53735/53735fig5.jpg" /> 그림 5 : 글리 칸에 대한 단축 프로토콜은 샘플 탈 아미 노화를 최소화하기 위해 개발 18hr 송곳니 MALDI 스펙트럼은 PNGase F 다이제스트에 비교 하였다. 존재비의 평균 차이는 10 % 각각 MD (2.2.3) 및 SPI (2.2.2) 프로토콜, 5 %였다. 확인 된 48 글리 칸 중 90 % 이하 1.5 배의 변화보다. 표시 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

A Quantitative Glycomics and Proteomics Combined Purification Strategy

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

양적 글리코 믹스와 단백질 체학 복합 정제 전략

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

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JoVE Journal

Chemistry

14.8K 조회수.  North Carolina State University. A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

비디오: A Quantitative Glycomics and Proteomics Combined Purification Strategy

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

질량 분석에 의해 - 겔 개선 환원 적 β-제거 포괄적 인 O가 연결된과에 대한 설포 글라 이코믹스

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

연구

화학

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

깊은 프로테옴 Isobaric 라벨, 광범위 한 액체 크로마토그래피, 질량 분석 및 소프트웨어 기반 정량화 하 여 프로 파일링

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

생화학

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

생물학

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<meta charset="utf8"></head><body>Glycomics-Guided Glycoproteomics를 사용한 대장암 조직의 프로파일링

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다. 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

양적 글리코 믹스와 단백질 체학 복합 정제 전략

요약

더 많은 비디오 탐색

이 비디오의 챕터

질량 분석에 의해 - 겔 개선 환원 적 β-제거 포괄적 인 O가 연결된과에 대한 설포 글라 이코믹스

깊은 프로테옴 Isobaric 라벨, 광범위 한 액체 크로마토그래피, 질량 분석 및 소프트웨어 기반 정량화 하 여 프로 파일링

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.