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.
在蛋白质组学领域,滤波器计算机辅助样品制备(FASP)已被广泛地用于其以最小化起始材料的量,降低样品制备的工件,并最大限度地提高样品通量1能力通过。然而,这样的方法还没有出现,并获得牵引力为糖组学的领域。高通量的发展,正在由于糖基化的生物防御的积分作用和由癌症或疾病2,3-其调制所需要定量的工作流程。在哺乳动物中,N- -glycans是由重复糖单元(己糖(十六进制),氨基己糖(HexNac),唾液酸(的NeuAc),和岩藻糖(Fuc)),其布置的芯结构(六角3 HexNac 2)4共价结合的以天冬酰胺。虽然当异构体的计数glycospace是相当大的(> 10 12),它是相当小的一个组合物的基础上和分子量典型地为1,000-8,000达5 <范围/ SUP>。类的组成均一性和聚糖的亲水性提出了独特的挑战,以净化,分 离,和质谱(MS)的工作流程6。
传统上,N- -glycans从蛋白质或肽通过肽Ñ消化糖苷酶F(PNG酶F),然后通过凝集素亲和层析7富集,由酰肼珠8捕获,或通过固相萃取(SPE)9,10纯化。虽然这些方法都是高度有效的,它们引入脱盐额外的步骤,限制同时处理的样本的数目。在过去的十年中,许多用于糖组学的高通量平台已经被提出。 Kim等人发表使用真空操作时,SPE 96孔板11的半自动化方法。或者,亲和过滤方法(N- -glyco-FASP)由曼基,其所需的网络连接的初始衍生开发滤波器与凝集素12的复合物。最后,托马斯-奥茨组提出了一个半定量的方法,辅助ñ-Glycan分离(毒牙)过滤器,它利用了glycospace 13的窄组成的大小。依靠分子量截止的过滤器,小的污染物被第一洗涤浪费,然后N个 -glycans消化和洗脱。去糖基化蛋白保留在过滤器上在本协议中,可以进行在线FASP。
鉴定并通过电喷雾电离聚糖的定量(ESI)MS需要离线分离的(部分)号决议,同分异构体和衍生的检测低丰度的物种。标记在个性与聚糖肼标记战略正火时赋予与反相液相层析(RPLC)14,15的兼容性。 4-苯乙基 – 苯甲酰肼(P2PGN)疏水标签介导的聚糖的亲水性,ENH由平均ancing电离,四倍16。在轻便条件1化学计量:虽然其他技术,如全甲基17或胺反应性标记化学18,提供了类似的优点,在酰肼反应,聚糖反应1。相对定量通过用天然(NAT)或13 的 C 6稳定的同位素标签(SIL)的衍生样品的串联分析来实现的。
下面的方法演变等离子应用和夫妇它的精确的相对定量P2GPN疏水标记的獠牙。此外,它的目的是在样品的单一等份执行射击枪蛋白质组学,脱酰胺剖析和定量糖组学,而不损害分析的完整性。
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 |