Forbedret i-gel Reduktive β-Elimination for Omfattende O-bundne og sulfo-Glykobiologi ved massespektrometri
Summary
In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive β-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.
Abstract
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 β-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.
Introduction
Glycosylering er et essentielt protein posttranslationel modifikation, der bidrager til organismal fysiologi, væv patologi og cellulær genkendelse 1-3. Til trods for store fremskridt i analytisk glycoscience, karakteriserer den komplette mangfoldighed glykaner på et specifikt protein fortsat er et særdeles udfordrende opgave, især på proteiner isoleret fra primære biologiske kilder. Ikke desto mindre, mikroheterogeniteten glycoproteinkomponenter glycaner ofte påvirker funktionelle interaktioner med andre proteiner. Derfor er væsentlige for forståelsen af fysiologiske betydning af cellulær og væv glycosylerings 4,5 karakterisering af glykan mangfoldighed. For at forstå bidrag glycoprotein glycosylering væv fysiologi og patofysiologi, robust, følsomme, og omfattende glycomic analytiske teknikker er blevet stadig vigtigere. I proteomanalyse er protein identifikationer generelt opnås ved LC-MS/ MS-analyse af tryptiske peptider 6. Proteinfordøjelsen kan udføres under anvendelse af et oprenset protein eller proteiner løst ved SDS-PAGE efter i-gel fordøjelse med proteaser, såsom trypsin 7-9. Pre-berigelse af proteinblandingen ved SDS-PAGE øger dybden og nøjagtigheden af protein ID. Udviklingen af analoge strategier for glycomic analyse af glycoprotein glycosylering ligger på forkant med glycoscience.
De to hovedklasser af glycaner er knyttet til protein backbone enten gennem N-binding eller O-binding. N-forbundne glycaner er bundet til asparagin (Asn) rester findes som en del af en sequon defineret som Asn-X-Ser / Thr / Cys (X er en vilkårlig aminosyre bortset fra prolin), og kan frigives ved enzymatisk fordøjelse med peptid-N -glycanase (PNGaseF eller A), enten i opløsning, i-gel eller på blot 10-12. O-bundne glykaner er hovedsagelig bundet til serin (Ser) eller threonin (Thr) -rester. Imidlertid har kun ét enzym blevet identificeret som er i stand til at frigive O-bundne glycaner fra glycoprotein, og det har en yderst begrænset glycan specificitet, frigiver kun de simpleste O-bundne glycaner. Kemisk frigivelse strategierne stadig den foretrukne metode for en omfattende frigivelse af O-bundne glycaner fra glycoproteiner. Reduktiv eller ikke-reduktiv β-elimination, eller hydrazinolyse er godt karakteriserede kemisk frigivelse teknikker og er i dag de mest anvendte metoder til at frigive O-bundne glycaner fra glycoproteiner 13,14. Selv reduktiv β-eliminering er blevet anvendt til at frigive O-bundne glycaner fra glycoproteiner adskilt ved SDS-PAGE, tidligere fremgangsmåder kræves HPLC-separation til efterfølgende analyse 15-17.
Multidimensional MS (MS n) analyse giver i øjeblikket den rigeste kilde til strukturelle data til karakterisering glycaner frigivet fra glycoproteiner isoleret i de beløb, der forventes fra de fleste biologiske kilder. Dybden af MS-baseret strukturel karakterisering lettes meget ved permethylating de frigivne glykaner forud for deres analyse. Permethylering forbedrer ionisering og har tendens til at udligne molære signalrespons på tværs af en bred vifte af glycanstrukturer 18,19. Derudover permethylering utvetydigt tags terminal- og monosaccharid dele med markante masserne og dermed forbedre den strukturelle belysning 20-23. For eksempel, er generelt svære at opdage, da ikke-permethyleret arter ved MS sure glykaner. Selvom sure glycaner kan detekteres i negativ ion-mode af MS, er det umuligt at detektere både sure og neutrale glycaner i samme ion-mode. En stor fordel ved glycan permethylering er, at alle de frie hydroxylgrupper (OH) på en glycan s monosaccharid substituenter vil blive afsluttet med en methylgruppe (OCH3 eller OMe), således en sialylerede glykan anklager neutraliseres, hvilket gør dem opdages som permethyleret neutral (AsienLO) glykaner. Men hydroxyler sulfatgrupper på sulfoglycans er resistente over for permethylering, hvilket resulterer i tilbageholdelse af anionisk ladning, der undertrykker ionisering og nedsætter følsomheden. Denne undertrykkelse forhindrer i øjeblikket omfattende glycomic analyse af meget komplekse glycoproteiner såsom muciner, som bærer en stor overflod af sulfaterede glykaner 24-26.
Nylige rapporter om rensende sulfaterede glykaner anvendte opladet, omvendt fase chromatografi at rense og separate permethyleret glykaner før MALDI analyse. Denne metode bygger på fuldstændig adskillelse af sulfaterede og ikke-sulfaterede glykaner ved hjælp af forskellige mobile faser for eluering, som vi har fundet at være lempeligere end organiske fase partitionering. Derfor er nye teknikker, der er egnede til påvisning og berigelse af sulfoglycans præsenteres her. Disse teknikker gør det muligt for kvantitativ genvinding af sulfaterede glycaner i den vandige fase efter vand: DCM (dichlormethan)ekstraktion, som rutinemæssigt udføres ved afslutningen af glycan permethylering reaktioner 27. Vigtigere er det, denne robuste adskillelse af permethylerede sulfoglycans fra en blanding af permethylerede non-sulfaterede glykaner samtidig beriger for ladede arter samtidig forenkle MS 2 fragmenteringsmønstre. En omfattende protokol for forbedret i-gel O-bundet glycan analyse præsenteres også. Den forbedrede protokol forbedrer glycan genopretning, øger den strukturelle information fås via MS n analyse af permethylerede glykaner og forbedrer følsomheden af sulfoglycomic analyser anvendes til væsentlige glycoproteiner isoleret fra biologiske kilder.
Denne protokol er beregnet til O-koblet glycan analyse af hele glycoprotein ekstrakter eller for et bestemt glycoprotein af interesse løses ved SDS-PAGE og er sammensat af tre eksperimentelle procedurer; A) gel oprydning, B) i-gel reduktiv β-elimination, og C) glycan permethyning. Målet er at opnå fuldstændige O-bundne glycomic data for glycoproteiner høstet fra primære kilder af biologisk interesse (figur 1). Glycoproteiner adskilt ved SDS-PAGE visualiseres ved farvning og bånd af interesse udskæres og den resulterende gel båndet skæres i små stykker. Gelstykkerne er affarvet og underkastet ethylacetat vasker for at fjerne forureninger gel (figur 2A). Glycan frigivelse opnås ved in-gel reduktiv β-eliminering (figur 2B), og de frigivne glycaner er permethyleret. Vandige organiske ekstraktion efter permethylering kvantitativt opdeler de anioniske sulfaterede glykaner væk fra ikke-sulfaterede neutrale glykaner (figur 2C). In-gel reduktiv β-eliminering koblet til vandige organiske ekstraktion muliggør karakteriseringen af O-bundne glycaner og sulfoglycans frigivet fra små mængder af glycoprotein separeret ved SDS-PAGE. Den strategisk overblik er summarized i figur 1 og detaljer er vist i figur 2. Desuden kan en del af affarvet og vasket gelstykker anvendes til protein ID ved standard LC-MS / MS proteomiske teknikker.
Protocol
Representative Results
Discussion
Combining in-gel reductive β-elimination with aqueous-organic extraction enhances the sensitivity and depth of structural data that can be acquired for characterizing sulfated and non-sulfated O-linked glycans harvested from small amounts of mucin-type glycoproteins resolved from other proteins by SDS-PAGE. The essential advances of the technical approaches presented in this study are: (a) facile removal of gel derived contaminants by simple washing steps; (b) quantitative recovery of permethylated sulfoglycans in t…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the grant P01HL107151 from the NHLBI/NIH. The authors also gratefully acknowledge the support and access to instrumentation provided through grant P41GM103490 from the NIGMS/NIH.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Water, deionized water | Sigma-Aldrich | 270733-4L | CHROMASOLV, for HPLC |
| Acetic acid, Glacial | Fisher | A38-500 | Certified ACS≥99.7 w/w % |
| Methanol | Sigma-Aldrich | 34860-4L-R | CHROMASOLV, for HPLC, ≥99.9% |
| Ammonium bicarbonate | Fluka | 09830-500G | BioUltra, ≥99.5% (T), Step 1 |
| Acetonitrile | Sigma-Aldrich | 34998-4L | CHROMASOLV Plus, for HPLC, ≥99.9%, Step 1 |
| Ethyl acetate | Fluka | 34972-1L-R | LC-MS CHROMASOLV, Step 1 |
| Sodium borohydride | Aldrich | 213462-25G | ReagentPlus, 99%, Step 2 |
| Iodomethane | Sigma-Aldrich | 289566-100G | ReagentPlus, 99.5%, Step 6. Store at 4 °C until use. Sit at room temperature before use. |
| Sodium hydroxide solution, 50% w/w | Fisher | SS254-1 | Certified, Step 6 |
| Dimethyl sulfoxide, anhydrous | Sigma-Aldrich | 276855-1L | Anhydrous, ≥99.9%, Step 6 |
| Methanol, anhydrous | Sigma-Aldrich | 322415-100ML | Anhydrous, 99.8%, Step 6 |
| Dichloromethane | Sigma-Aldrich | 34856-4L | CHROMASOL®, for HPLC, ≥99.8%, contains amylene as stabilizer, Step 7 |
| Dowex 50WX8 hydrogen formhydrogen form 100-200 mesh | Sigma-Aldrich | 217506-500G | Step 3 |
| AG 50W-X8 Resin | Bio-Rad | 142-1441 | Step 3 |
| BAKERBOND spe 1 ml x 100 mg Solid Phase Extraction Column, PP, Octadecyl (C18) Reverse Phase | JT Baker | JT-7020-01 | Step 5 and 8 |
| 7.5% Mini-PROTEAN TGX Precast Gel | Bio-Rad | Step 1 | |
| Bio-Safe Coomassie Stain | Bio-Rad | 161-0786 | G-250, Step 1 |
| Silver Stain Kit for Mass Spectrometry | Pierce | 24600 | Step 1 |
| Oligosaccharides Kit (Maltotriose, Dp3: R474140) | Supelco | 47265 | |
| Oligosaccharides Kit (Maltotetraose, Dp4: R474135) | Supelco | 47265 | |
| Disposable Pasteur Pipets, Glass, Short Tip | VWR | 14673-010 | Wash before use |
| PYREX 13 x 100 mm Disposable Round Bottom Threaded Culture Tubes | Corning | 99447-13 | Wash before use |
| PYREX 16 x 125 mm Disposable Round Bottom Threaded Culture Tubes | Corning | 99447-16 | Wash before use |
| Phenolic Caps/Closures with PTFE-Faced Rubber Liner | Kimble | 45066C-13 | Wash before use |
| Phenolic Caps/Closures with PTFE-Faced Rubber Liner | Kimble | 45066C-15 | Wash before use |
| Hamilton HPLC syringe | HAMILTON | 81265 | volume 500 μL, needle size 22 ga |
| Hamilton HPLC syringe | HAMILTON | 81165 | volume 250 μL, needle size 22 ga |
| Hamilton HPLC syringe | HAMILTON | 81065 | volume 100 μL, needle size 22s ga |
| Hamilton HPLC syringe | HAMILTON | 80965 | volume 50 μL, needle size 22s ga |
| Hamilton Calibrated Syringes | HAMILTON | 80300 | volume 10 μL, needle size 26s ga |
| Petri Dish Glass 100mm x 15mm | GSC INTERNATIONAL INC | 1500-4 | Wash before use, Step 1 |
| Bard-Parker Surgical Blades | Fisher | 371310 | Step 1 |
| Reacti-Therm Heating/Stirring Module | Pierce | 18870 | Step 1, 4 and 7 |
| Heating Blocks | Fisher | 125D | Step 2 |
| Pyrex fiber glass wool borosilicate pore size 8 μm | Aldrich | CLS3950 | Step 3 |
| Fused Silica CutterTubing cutter | alltech | 3194 | Step 3 |
| Multi-tube vortexers | VWR | 444-7063 | Step 6 |
| Lyophilizer 25EL Freezemobile | Virtis | 25EL | |
| Centrifuge | VWR | Clinical 50 | |
| LTQ Orbitrap Discovery | Thermo Fisher Scientific | 0 |
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