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.
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.
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 (> 10 ~ 12)은, 그 조성에 기초하여 매우 작고, 전형적으로는 분자량 1,000-8,000 다 5 <에서부터/ SUP>. 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.
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 β-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.
The authors have nothing to disclose.
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.
Acetic Acid (50%): | Sigma Aldrich | 45754 | |
Acetonitrile, HPLC grade | Burdick & Jackson | AH015-4 | |
Ammonium Bicarbonate | Sigma Aldrich | A6141 | |
Bradford Reagent | Sigma Aldrich | B6916 | Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1) |
Calcium chloride | Sigma Aldrich | C1016 | |
Centrifuge | Eppendorf | 5804 R | Alternate centrifuges that reach 14,000 x g are suitable |
DL-Dithiothreitol, 1M in solution | Sigma Aldrich | 646563 | |
Easy-nLC 1000 | Thermo Scientific | LC120 | Alternate nano or ultra high pressure LCs will produce similar data, such as: 1. Dionex UltiMateÒ 3000 LC (Thermo Scientific) 2. Acquity UPLC (Waters) |
Floating Tube Rack | TedPella | 20831-20 | |
Fetuin | New England Biolabs | P6042S | |
Fisher Scientific Isotemp Standard Lab Ovens | Fisher Scientific | 11-690-625F | Alternate incubators that reach 56 °C are suitable |
Formic Acid | Sigma Aldrich | 56302 | |
GE Microwave Oven | General Electric | 57B5 E82904 | Any microwave with adjustable power settings is suitable |
INLIGHT Glycan Tagging Kit | Cambridge Isotope Laboratories | GTK-1000 | The INLIGHT kit provides NAT and SIL versions of the P2GPN reagent. |
Iodoacetamide | Sigma Aldrich | A3221 | |
Kinetix 2.6 mM, 100 Å, C18 bulk stationary phase | Phenomenex | Bulk Media | Alternative: Any C18 stationary phase £ 5 mM |
Mascot Daemon Software and Server | Matrix Science | Alternative: Proteome Discoverer Software (Thermo Scientific) | |
Methanol, HPLC grade | Burdick & Jackson | AH230-4 | |
PicoFrit Self-Pack Column: 360 um, OD 75um ID, 15 um tip, non-coated, 5 per box, 50 cm | New Objective | 1 5 PF360-75-15-N-5 | |
PNGase F (glycerol-free), 75,000 units/ml | New England BioLabs | P0705L | |
Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer | Thermo Scientific | Alternate high mass accuracy (£ 5 ppm) mass spectrometers will provide similar data | |
RNase B | New England Biolabs | P7817S | |
Trypsin from Porcine Pancreas | Sigma Aldrich | T6567-5X | |
Urea | Sigma Aldrich | 51456 | |
Vacuum ConcentratorSavant SPD131DDA SpeedVac Concentrator | Thermo Scientific | SPD131DDA | Alternate vacuum concentrators are suitable |
Vivacon 500 30 kDa Filters | Sartorius Stedim Biotech | VN01H22 | Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10 kDa Membrane (Millipore UFC501096) |
Water, HPLC grade | Burdick & Jackson | AH365-4 | |
Water, 18O | Cambridge Isotope Laboratories | OLM-240-97-1 | The addition of 18O in the PNGase F digest step is optional and may not be necessary for deamidation studies completed with 95% confidence |
Xcalibur 2.0 | Thermo Scientific | XCALIBUR20 | |
Zwittergent Test Kit | Merck Millipore | 693030 |