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.
I feltet av proteomikk, har filter hjulpet prøvepreparering (FASP) vært i omfattende bruk for dens evne til å minimalisere mengden av utgangsmaterialet, redusere prøveopparbeidelse gjenstander, og maksimere en prøvekapasitet. Imidlertid har en slik fremgangsmåte, men å dukke opp og få trekkraft for feltet glycomics. Utvikling av high-throughput kvantitative arbeidsflyter nødvendig på grunn av den integrerte rolle glykosylering i biologisk forsvar og dens modulering av kreft eller sykdommer 2,3. I pattedyr, er N -glycans består av repeterende sakkaridenheter (heksoser (Hex), hexosamines (HexNac), sialinsyrer (NeuAc), og fucoses (FUC)), som dekorerer en kjernestruktur (Hex 3 HexNac 2) 4 kovalent bundet til asparagin. Selv om glycospace er betraktelig stor når isomerer telles (> 10 12), er det ganske liten på en sammensetning basis og molekylvekter typisk i området fra 1,000-8,000 Da 5 </ Sup>. Den kompositoriske homogenitet av klassen og hydrophilicity av glykaner utgjøre unike utfordringer til rensing, separasjon, og massespektrometri (MS) arbeidsflyt 6.
Tradisjonelt er N -glycans fordøyd fra proteiner eller peptider av peptid N -glycosidase F (PNGase F) og deretter beriket av lektin affinitetskromatografi 7, fanget av hydrazide perler 8, eller renset via fast fase ekstraksjon (SPE) 9,10. Selv om disse metodene er alle meget effektive innføre de ekstra trinn for avsalting og begrense antall prøver samtidig behandlet. I løpet av det siste tiåret, har en rekke high-throughput plattformer for glycomics blitt foreslått. Kim et al., Publisert en semi-automatisert fremgangsmåte ved bruk av en vakuum-operert, SPE 96-brønns plate 11. Alternativt ble en affinitet-filter-metoden (N -glyco-FASP) som er utviklet ved hjelp av Mann-gruppen, som kreves for den første derivatisering av den fi-lter med en sammensetning av lektiner 12. Til slutt, Thomas-Oates Gruppen foreslo en semi-kvantitativ metode, Filter Aided N -Glycan separasjon (FANGS), som utnyttet den smale sammensetning størrelsen på glycospace 13. Avhengig av molekylvekt cut-off-filter, ble små forurensninger først vasket til avfall, og deretter N–glycans ble spaltet og eluert. Deglykosylert proteiner forblir på filteret i denne protokollen, og kan utsettes for på-linje FASP.
Identifikasjon og kvantifisering av glykaner ved elektrospray ionisering (ESI) MS krever off-line separasjon av (delvis) oppløsning av isomerer og derivatisering for deteksjon av lav-artene. Merking i individualitet når normal med sukker hydrazide tags strategi overfører kompatibilitet med revers-fase væskekromatografi (RPLC) 14,15. Den 4-fenetyl-benzohydrazide (P2PGN) hydrofob tag formidler hydrophilicity av glykaner, ENHsering ionisering av i gjennomsnitt fire ganger 16. Selv om andre teknikker så som permethylation 17 eller amin-reaktive kjemikalier tagging 18, gir tilsvarende fordeler, i hydrazidet reaksjonen er glykaner omsettes 1: 1 støkiometrisk i lettvinte forhold. Relativ kvantifisering oppnås ved tandem analyse av prøver derivatisert med native (NAT) eller 13 C 6 stabile isotopen etiketter (SIL).
Følgende metode utvikler FANGS for plasma applikasjoner og par den til P2GPN hydrofobe tag for nøyaktig relativ kvantifisering. Videre ble det utformet for å utføre shot-gun proteomikk, deamidering profilering og kvantitative glycomics på en enkelt porsjon av prøven, uten å kompromittere integriteten av analysene.
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 |