والكمي Glycomics والبروتيوميات المشتركة الاستراتيجية تنقية
Summary
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.
Abstract
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.
Introduction
في مجال البروتينات، وقد اعتمدت على نطاق واسع مرشح بمساعدة إعداد العينات (FASP) لقدرته على تقليل كمية من المواد ابتداء، والحد من القطع الأثرية وإعداد العينات، وتحقيق أقصى قدر من الإنتاجية عينة 1. ومع ذلك، فإن مثل هذا الأسلوب حتى باتت وكسب الجر مجال glycomics. تطوير الإنتاجية العالية، وهناك حاجة سير العمل الكمية نظرا للدور لا يتجزأ من بالغليكوزيل في الدفاع البيولوجي وتعديل من قبل السرطان أو أمراض 2،3. في الثدييات، وتتكون -glycans N من تكرار وحدة السكريد (hexoses (عرافة)، hexosamines (HexNac)، والأحماض اللعابي (NeuAc)، وfucoses (الجبهة المتحدة))، التي تزين بنية الأساسية (عرافة 3 HexNac 2) 4 ملزمة تساهميا لالهليونين. على الرغم من أن glycospace هو كبير إلى حد كبير عندما تحسب أيزومرات (> 10 12)، أنها صغيرة جدا على أساس تكوين والأوزان الجزيئية تتراوح عادة من 1،000-8،000 دا 5 </ سوب>. التجانس التركيبي للطبقة وhydrophilicity من glycans تحديات فريدة لتنقية وفصل، ومطياف الكتلة (MS) سير العمل 6.
تقليديا، يتم هضمها N -glycans من البروتينات أو الببتيدات التي كتبها peptide- N -glycosidase F (PNGase F) ومن ثم إثراء كتين تقارب اللوني 7، استولت عليها الخرز هيدرازيد 8، أو تنقيته عن طريق استخراج المرحلة الصلبة (SPE) 9،10. في حين أن هذه الأساليب كلها فعالة للغاية، وتقديم خطوات إضافية لتحلية والحد من عدد من عينات معالجة في وقت واحد. على مدى العقد الماضي، وقد تم اقتراح عدد من منصات عالية الإنتاجية لglycomics. كيم وآخرون نشرت طريقة شبه الآلي باستخدام تعمل فراغ، لوحة SPE 96-جيدا 11. بدلا من ذلك، تم تطوير وسيلة تقارب فلتر (N–glyco FASP) من قبل مجموعة مان، الذي يتطلب اشتقاق الأولي للفايlter مع مزيج مركب من يكتينس 12. وأخيرا، اقترح الفريق توماس أوتس طريقة شبه الكمية، وتصفية بمساعدة N الفصل -Glycan (الأنياب)، التي استغلت حجم التكوين الضيق للglycospace 13. الاعتماد على الوزن الجزيئي قطع المرشحات، وجرفت المياه الملوثة صغيرة أولا أن تضيع ثم تم هضم -glycans N ومزال. تبقى البروتينات Deglycosylated على مرشح في هذا البروتوكول، ويمكن أن تخضع لFASP في الخط.
تحديد وتقدير من glycans بواسطة تأين بالإرذاذ الإلكتروني (ESI) MS يتطلب خارج خط فصل للقرار (جزئي) من سداسي واشتقاق للكشف عن الأنواع المنخفضة للوفيرة. وضع العلامات في الفردانية عندما تطبيع مع استراتيجية علامات هيدرازيد غليكان يمنح التوافق مع اللوني العكسية مرحلة السائل (RPLC) 14،15. 4-phenethyl-benzohydrazide (P2PGN) العلامة مسعور تتوسط في hydrophilicity من glycans، ENHancing التأين التي، في المتوسط، أربعة أضعاف (16). على الرغم من التقنيات الأخرى، مثل permethylation 17 أو أمين رد الفعل كيمياء علامات 18، توفر مزايا مماثلة، في رد فعل هيدرازيد، وكان رد فعل glycans 1: 1 stoichiometrically في الظروف السهلة. ويتحقق الكمي النسبي عن طريق تحليل جنبا إلى جنب من عينات derivatized مع الأم (NAT) أو 13 C 6 تسميات مستقر النظائر (سيل).
الأسلوب التالي تطور الأنياب لتطبيقات البلازما والأزواج إلى العلامة P2GPN مسعور لتقدير نسبي دقيقة. وعلاوة على ذلك، تم تصميمه لأداء البروتينات طلقة بندقية، والتنميط نزع الأميد، وglycomics الكمية على قسامة واحدة من العينة، دون المساس بسلامة التحليلات.
Protocol
Representative Results
Discussion
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.
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
References
- Wisniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. Universal sample preparation method for proteome analysis. Nat. Meth. 6, 359-362 (2009).
- Preston, R. J. S., Rawley, O., Gleeson, E. M., O’Donnell, J. S. Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities. Blood. 121, 3801-3810 (2013).
- Hasnain, S. Z., Gallagher, A. L., Grencis, R. K., Thornton, D. J. A new role for mucins in immunity: Insights from gastrointestinal nematode infection. Int. J. Biochem. Cell Biol. 45, 364-374 (2013).
- Roseman, S. Reflections on glycobiology. J. Biol. Chem. 276, 41527-41542 (2001).
- Laine, R. A. A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: The isomer barrier to development of single-method saccharide sequencing or synthesis systems. Glycobiology. 4, 759-767 (1994).
- Zaia, J. Mass spectrometry and glycomics. Omics. 14, 401-418 (2010).
- Hirabayashi, J. Lectin-based structural glycomics: Glycoproteomics and glycan profiling. Glycoconjugate J. 21, 35-40 (2004).
- Nilsson, J., et al. Enrichment of glycopeptides for glycan structure and attachment site identification. Nat. Methods. 6, 809-811 (2009).
- Redmond, J. W., Packer, N. H. The use of solid-phase extraction with graphitised carbon for the fractionation and purification of sugars. Carbohyd. Res. 319, 74-79 (1999).
- Ruhaak, L. R., et al. Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins. Anal. Chem. 80, 6119-6126 (2008).
- Kim, Y. -. G., et al. Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform. Anal. Biochem. 391, 151-153 (2009).
- Zielinska, D. F., Gnad, F., Wiśniewski, J. R., Mann, M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell. 141, 897-907 (2010).
- Abdul Rahman, S., et al. Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling. J. Proteome Res. 13, 1167-1176 (2014).
- Walker, S. H., Carlisle, B. C., Muddiman, D. C. Systematic comparison of reverse phase and hydrophilic interaction liquid chromatography platforms for the analysis of N-linked glycans. Anal. Chem. 84, 8198-8206 (2012).
- Walker, S. H., Taylor, A. D., Muddiman, D. C. Individuality normalization when labeling with isotopic glycan hydrazide tags (INLIGHT): A novel glycan-relative quantification strategy. J. Am. Soc. Mass Spectr. 24, 1376-1384 (2013).
- Walker, S. H., Lilley, L. M., Enamorado, M. F., Comins, D. L., Muddiman, D. C. Hydrophobic derivatization of N-linked glycans for increased ion abundance in electrospray ionization mass spectrometry. J. Am. Soc. Mass. Spectr. 22, 1309-1317 (2011).
- Baldwin, M. A., et al. Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: Analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein. Anal. Biochem. 191, 174-182 (1990).
- Harvey, D. J. Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry. J. Chromatogr. B. 879, 1196-1225 (2011).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).
- Smith, P. K., et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85 (1985).
- Hecht, E. S., McCord, J. P., Muddiman, D. C. Definitive screening design optimization of mass spectrometry parameters for sensitive comparison of filter and solid phase extraction purified, inlight plasma N-glycans. Anal. Chem. 87, 7305-7312 (2015).
- Nepomuceno, A. I., Gibson, R. J., Randall, S. M., Muddiman, D. C. Accurate identification of deamidated peptides in global proteomics using a quadrupole orbitrap mass spectrometer. J. Proteome Res. 13, 777-785 (2013).
- Yen, T. -. Y., et al. Overcoming challenges and opening new opportunities in glycoproteomics. Biomolecules. 3, 270-286 (2013).
- Meiring, H. D., van der Heeft, E., ten Hove, G. J., de Jong, A. P. J. M. Nanoscale LC-MS(n): technical design and applications to peptide and protein. J. Sep. Sci. 25, 557-568 (2002).
- Apweiler, R., et al. UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 32, 115-119 (2004).
- Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 20, 3551-3567 (1999).
- Eng, J. K., McCormack, A. L., Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 5, 976-989 (1994).
- Gonzalez-Galarza, F. F., et al. A critical appraisal of techniques, software packages, and standards for quantitative proteomic analysis. Omics. 16, 431-442 (2012).
- Spivak, M., Weston, J., Bottou, L., Käll, L., Noble, W. S. Improvements to the Percolator Algorithm for Peptide Identification from Shotgun Proteomics Data Sets. J Proteome Res. 8, 3737-3745 (2009).
- Collier, T. S., et al. Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression. Rapid Commun Mass Sp. 25, 2524-2532 (2011).
- Kronewitter, S. R., et al. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome. Proteomics. 9, 2986-2994 (2009).
- Sheeley, D. M., Reinhold, V. N. Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole ion trap mass spectrometer: Neutral oligosaccharides and N-linked glycans. Anal. Chem. 70, 3053-3059 (1998).
