© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. It begins with the the with begins It biosynthesis. the non-template-driven is of which a consequence on any glycoprotein, present structures N-glycan of diversity ent state of the art proteomics. Complicating the analysis is the inher with can be that integrated glycosylation N-linked of for analysis tions of glycoproteins, there is increasing need for robust methods needed. is N-glycosylation of consistency the and intercellular recognition and host–pathogen interactions host–pathogen and recognition intercellular and and and IAVI Neutralizing Antibody Center, and Immunology Microbiology,of Department The ResearchScripps Institute, La Jolla, California, USA. containing sialic acid sialic containing structures complex-type processed fully require others whereas epitopes, in their glycans high-mannose underprocessed glycoprotein include envelope HIV glycosylated highly the of (bnAbs) Published online 3 May 2018; Massachusetts, USA. Correspondence should be addressed to J.C.P. ( of the proteins themselves proteins the of roles and have They in stability the critical conformation folding, proteins. of modifications post-translational mon and important (N-glycans) are among Asparagine-linked-glycans the most com I takes ~7 d to complete. T glycosylationsite-specific of glycoproteins is achieved, with up to thousands of spectra high-confidence hits for each glycosite. analysis; and (v) data analysis for and identification quantitation of peptides for the three glycosylation states. Full coverage of two endoglycosidases, nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 degree of glycan occupancy at each glycosite and the proportion of this analytical challenge, we developed a robust semiquantitative mass spectrometry (M multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. addition of to monosaccharides generate complex-type and hybrid glycans. glycan precursor to the sequon N 1 Liwei Cao N-glycan processing Global site-specific analysis of glycoprotein 1196 Claire M Delahunty proline). Although none of the proteins analyzed in the present present the in analyzed proteins the of none Although proline). except residue acid amino any be can X (where Asn-X-Thr/Ser glycosite, to a (OST) sequence-defined an by oligosaccharyltransferase polypeptide nascent the to donor glycosyl lipid-linked a Biochemistry and Cell Biology,Biochemistry Geisel MedicineSchool of at Dartmouth, Hanover, New Hampshire, USA. in in both mice and humans cans have been reported to occur during breast cancer gly progression high-mannose-type underprocessed of levels increased ple, the activity and pharmacodynamics of glycoprotein biothera peutics glycoprotein of pharmacodynamics and activity the growth conditions growth diseases, such as cancer many with has associated been N-glycans of biosynthesis Altered Department of Molecular of Medicine,Department The ResearchScripps Institute, La Jolla, California, USA. NTRO he he protocol can be performed by an experienced technician or with student/postdoc basic skills for proteomics experiments and protocol -glycans contribute to the folding, stability and functions of the proteins they decorate. T Given the importance of N-glycans to the structure and func and structure the to N-glycans of importance the Given | VOL.13 NO.6VOL.13 D 1 6 UCT , careful choice of cell lines used to express proteins express to used lines cell of choice careful , 1 I , 2 ON , Jolene K Diedrich | 2018 1 en bloc en 7 , and purification methods purification and , 11,1 5, E 1 doi:10.1038/n | ndo ndo H and , Jason S McLellan 6

5 , influenza 6 natureprotocols . As the structures of N-glycans affect affect N-glycans of structures the As . . Some broadly neutralizing antibodies . antibodies Some broadly neutralizing 1, transfer of Glc of transfer 2 , and participate as ligands in intra- intra- in ligands as participate , and A sn-X- PN prot.2018.02 7– © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 1 , Yuanhui Ma 9 Gase Gase F, to create unique mass signatures for the three glycosylation states; (iv) T , and AIDS hr/ S 3 er, followed by enzymatic trimming to a corehigh-mannose-type and sequential Man 3 4 , Dennis R Burton 9 18–2 10–1 GlcNAc 1 4 0 [email protected] . For exam to control control to , Nianshuang Wang 2 from from 3, 1 0 4 ------, . 2 N sites have been verified in previous studies by the presence of of presence the by glycans on glycopeptides intact studies previous in verified been have sites site (N-X-Cys/Val), glycosylation study an contains atypical these generated by both matrix-assisted laser desorption/ionization desorption/ionization laser N-glycans matrix-assisted both derivatized by of generated spectra mass principles. tandem biosynthetic Furthermore, with consistent glycan or high-mannose complex-type a as annotated is which ion, molecular each to to the Manconserved trimming further then glycan, high-mannose-type a to residues glucose the of removal by trimming to subjected then is glycan by analysis of glycoforms using MS using glycoforms of analysis by followed protein, the from glycans the release to A, PNGase and such as ery.F PNGase employ endoglycosidases, methods Several that are recognized and used by the cellular glycosylation machin to emerged have characterize N-glycans of glycoproteins and strategies identify the glycosites several decades, two past the Over glycoproteins of N-glycans Analysis of highly related set of complex-type structures ( structures complex-type of set related highly that produce a ars glycosyltransferases of by action the sequential vatization glycoprotein liquid chromatography (HPLC) chromatography liquid and highly processed complex-type glycans at other sites are are sites other at IgM glycans complex-type processed highly glycosites, and more or one at glycans high-mannose-type contain that proteins of examples Indeed, documented well ing enzymes. process the to glycans the of access the on based differ may site for glycoproteins with more than one glycosite, processing at each , 4 -glycans processed from high-mannose type to complex type. , John R Yates ). 23,2 2 Center for HIV/AIDS Vaccine and Immunology Immunogen Discovery 4 T 3 , influenza hemagglutinin influenza , his his process, mediated by the concerted action of , Matthias Pauthner 3 8 . The MS-based methods provide a composition for for composition a provide methods MS-based The . 10,27–2 4 Ragon Ragon Institute MGH,of MIT and Harvard, Cambridge, 1

9 . S

& James C Paulson 3 )-based )-based method that determines the GlcNAc hey are produced by transfer of the µ g g of protein. Here, we provide 2 21,2 core, followed by of sug addition 2 36,3 , Sung-Kyu Robin Park 2 . As in illustrated 8,25,2 7 with or without prior deri prior without or with 30–3 6 , and the HIV envelope envelope HIV the and , 5 or high-performance high-performance or

1 , 2 3 Department of of Department

Fig. Fig.

LC 1 Figure Figure ). Moreover, -M T o o address S /M S 1 1 , , the - - - -

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. endoglycosidase (e.g., PNGase F) that, in the process, converts converts process, the in that, F) PNGase (e.g., an endoglycosidase with glycan bound the from released are beads. peptides the Then hydrazide-activated to glycopeptides coupling by periodate-treated or lectins using by glycopeptides immobilize to is is performed in H in performed is the Asn-X-Thr/Ser sequence to Asp-X-Thr/Ser. When the reaction N-glycans expressed in different biological systems biological different in expressed of N-glycans information structural detailed of characterization for used MS (ESI) have ionization been widely and electrospray (MALDI) glucose; GlcNAc, N-acetylglucosamine; Man, mannose; Sia, sialic acid. Asn, asparagine; ER, endoplasmic reticulum; Fuc, fucose; Gal, galactose; Glc, mannose residues, which is required for recognition by Endo H (red oval). box; these include high-mannose and hybrid glycans that have two terminal Golgi apparatus. N-glycans that are Endo H-sensitive are shown in the red Figure 1 combined masses of the glycopeptides and N-glycan and glycopeptides the of masses combined deter and on based the are at present each glycosite glycoforms which mine glycopeptides intact analyze that methods MS/MS glycosylated. are cal systems, such as cells whole or tissues, to proteins that identify biologi complex surveying for useful These particularly glycosylated. are are methods that sites the of identification positive provides peptides eluted the of analysis MS/MS +3. Then of Asp to which the site is occupied by a glycan a by occupied is site the which to degree the and OST the by utilized are that glycosites the of tion occupied. is glycosite each which to extent the or utilized are glycosites which reveal not do they abundance, relative their on present a and the the of about glycoforms nature glycoprotein toolbox.org available software tools such as GRITS Toolbox ( proteins glyco serum of N-glycans in differences individual detecting or highly studied glycoproteins such as immunoglobulins (e.g., IgG), of glycosylation in characterizing utility wide find Such methods analysis can provide additional support for structure assignments. permethylation and/or digestions glycosidase using experiments supporting methods, both With species. glycan the of tification iden for standards N-glycan of times retention on rely methods manner. HPLC The in a data high-throughput glycomics archive Recently, there have been major advances in glycoproteomics glycoproteomics in advances major been have there Recently, A number of proteomics-based methods focus on identifica on focus methods proteomics-based of number A o-P-Asn- Dol-PP Man GlcNAc Gal Sia Glc Fuc

| 36,37,3 N-linked glycan processing in the endoplasmic reticulum and / ER ) are able to automatically process, annotate, and and annotate, process, automatically to able are ) 9 Endo H-sensitive . these Although methods provide key information An An -Asn- -Asn- -Asn- 2 O 18 , it creates a mass difference from Asn to to Asn from difference mass a creates it , -Asn- -Asn- © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. An An An -Asn- -Asn- -Asn- -Asn- 40–4 Golgi 4 -Asn- . The basic strategy strategy basic .The -Asn- http://www 30,3 8,28,29,45–4 4 . Freely Freely . -Asn- -Asn- .grits- 8 - - - - - . ionization efficiencies during MS analysis during efficiencies ionization Asn (Endo H–treated), or Asp (PNGase F–treated), display similar GlcNAc- Asn with (unoccupied), (Asn-X-Thr/Ser) glycosites ing Asn to Asp, with a +3 addition to the peptide mass. Peptides carry enriched prior to analysis, peptides with no glycan are lost, and and lost, are glycan no with peptides analysis, to prior enriched system immune the by attack against protect to shield glycan a creating N-glycans, 75–90 contains that (Env) trimer glycoprotein lope is for a the HIV enve vaccine candidate the primary that the fact from stems need The type. complex to type high-mannose from processed were glycans which to degree the assess (ii) and cosite gly each at N-glycans of occupancy the establish (i) would: that methods glycoproteomics for semiquantitative robust effort cine vac HIV the of from need the here arose described protocol The the Development protocol of the method and of applications assessed. be cannot occupancy glycosite of degree the to HIV Env do occur in 10–30% of HIV-1-infected patients HIV-1-infected of 10–30% in occur do Env HIV to of intact glycopeptides intact of peptides with large glycans, impedes comprehensive identification for dif fragmentations backbone as peptide good well obtaining in ficulties as site, glycosylation each at glycoforms of geneity hetero inherent the (ETD), dissociation electron-transfer (HCD), and dissociation collision high-energy (CID), dissociation collision-induced including methods, fragmentation different of glycans type type glycans, and complex-type glycans, as illustrated in high-mannose/hybrid- glycan, no carry that glycosites for tures signa mass unique to introduce treatments two endoglycosidase glycoprotein. any to applicable broadly is method raphy (HILIC) raphy purification methods, such as hydrophilic interaction chromatog different to with prior MS analysis digests from peptide enriched analysis the inherent as low ionization efficiency of glycopeptides during MS well as mixture, digest protein the in glycopeptides of dance abun low relatively the are approach this of limitations Major hleg de o nnw inzto efcece fr pep structures for acid- containing sialic with efficiencies those for especially glycoform, ionization each with tides unknown to due challenge the glycan detected of structures at each glycosite is still a substantial abundance relative the of assessment quantitative However, ( Byonic as such experiments is possible by using commercial algorithms such three glycosylation states at each glycosite detected glycosite each at states glycosylation three the of distribution the relative area to peak quantify ion intensity distinguishing feature of the method is the use of multiple multiple of use the is method the of feature distinguishing glycosite on the HIV Env HIV the on glycosite each at N-glycans complex-type and high-mannose-type of tion Env vaccine, we developed a method that could assess the propor glycans high-mannose with interactions show bnAbs some Importantly, presence of H of presence are F PNGase removed in with N-glycans complex the remaining Asn residue that adds +203 to the peptide mass. Subsequently, the at least 5 mannose residues ( have that N-glycans hybrid-type and high-mannose- all remove to used is H) (Endo H endoglycosidase digestion, protease After One of the key aspects of our method is the sequential use of of use sequential the is method our of aspects key the of One 53,5 12,15,59,6 57,5 40,41,49,5 4 8 . Despite the dense cover of N-glycans, bnAbs that bind , whereas others show dependence on complex-type complex-type on dependence show others whereas , http:// 2 29,51,5 O 0 0 . Thus, to support the rational design of an HIV HIV an of design rational the support to .Thus, . For these reasons, glycopeptides are typically typically are glycopeptides reasons, these For . 18 www.proteinmetrics.c , which both removes the glycan and converts converts and glycan the removes both , which 2 and hydrazide chemistry and hydrazide natureprotocols 28,3 3 8 . Moreover, if the glycopeptides are are glycopeptides the if Moreover, . 1 0 0 . Annotation of LC-MS/MS data from data LC-MS/MS .of Annotation . However, as demonstrated here, the the here, .However,demonstrated as Fig. 1 ). This enzyme leaves a GlcNAc-

| VOL.13 NO.6VOL.13 om/products/byonic 10,4 5 , allowing ,us to use allowing 4 0 protocol . Despite . the use Despite 10,6 | 2018 1 Figure 2 . Another . Another |

55,5 1197 / a ). 6 ------. .

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. of the protocol. occupied by high-mannose/hybrid or complex-type glycans. ( mass signatures for peptide glycosites that are not occupied, or that are sylation site sylation into the site occupancy and processing of N-glycans at each glyco manner. high-throughput a in data analyze to software proteomics robust of use the allowing analysis, teomics effectively strategy converts the glycoproteomics analysis to a pro simple This coverage. sequence in >95% resulting glycosite, each for hits spectra of up to thousands generate can which proteases, Figure 2 1198 only only 30 states, and a can glycosylation be analysis on complete conducted to survey glycosites in more complex samples such as cells or or cells as such serum. human samples complex more in glycosites survey needed to be would protocol the of modification section, design’ in the However,‘Experimental proteins. as described 10–12 only comprise that coronavirus) virus, HIV,influenza (e.g., viruses such protein samples, to as membrane-enveloped more-complex applicable principle, in is, it glycoproteins, purified of analysis glycosylation monosaccharides. site-specific for is designed protocol this Although between linkages structure glycosidic with precise the complete provides method neither However, ing. process glycan and occupancy site on information quantitative semi provides which protocol, this to complementary are and mation on the spectra of glycans at individual glycosites individual at infor glycans of more spectra the on provide mation glycopeptides intact methods of MS/MS analysis on based endoglycosidases. the of specificities the by removed before LC-MS/MS, and the class of the glycan is inferred typically have low abundance low have typically structures H.However, Endo hybrid by cleaved are they because acid (see sialic contain can potentially which structures, hybrid that noted be should It monomer. per glycosites 30 to up Env,comprising Complex-type High-mannose a protocol A major advantage of this method is that it provides a glimpse a glimpse provides it that is method this of advantage A major One One major limitation of the method is that glycan structures are (hybrid) glycan No

| VOL.13 NO.6VOL.13

µ | Proteases ppie)EndoH (peptides) Schematic overview of the protocol. ( Fig. Fig. g g of protein, even for complex glycoproteins such as HIV N N N N 1 0 1 a . It provides a semiquantitative analysis of the of three .analysis It a provides semiquantitative Treatment adapted from ref. ), are included in the high-mannose-type category category ), are in included the high-mannose-type N N | 2018 PNGase F (O 18 | D N N

H natureprotocols 2 O) Predicted 29,4 1 mass +203 MS +3 +0 0 , Nature Publishing Group. 8 . © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. b Denaturation andalkylationofglycoprotein a combinationoftrypsinandchymotrypsin Sequential endoglycosidasetreatment: a use peptideswithpeakarea>5×10 assessment of ) Introduction of novel Buffer exchangeforglycoproteins sum peakareaforeachpeptide, identification ofallpeptides,and Endo HfollowedbyPNGaseF triple digestion,chymotrypsin Denaturation ofproteases Converttopeakarea: Protease treatments: LC-MS/MS analysis Analysis ofdata: (Steps 26–35) (Steps 19–25) (Steps 13–17) (Step 50–52) (Step 37–49) (Steps 1–12) (Step 36) (Step 18) N +0, b ) The workflow N +3, and 27–29,6 N +203 8 2 - - - - - ,

pcfc lcslto aayi o prfe glycoproteins purified of analysis glycosylation specific site- in progress substantial been has there decade past the Over Alternative methods different glycoforms different many of mixture a actually is glycopeptide each that fact the to in part and glycopeptides of efficiencies low ionization relatively the to part in attributed is which methods, their for required be may material of quantities However, glycosite. milligram each at glycoforms individual about information complementary pro can vide thus and analysis, MS to prior glycopeptides of ment enrich without or with glycopeptides intact characterizing on 45,48,62–6 groups Cripsin and Desaire the by Env HIV-1 on work the is protocol, have long been in use in been long have analysis. MS to prior glycopeptides of enrichment and/or glycopeptides, corresponding the and different peptides between markedly efficiencies to ionization due occupancy, site about information limited is Moreover,there analysis. MS during species N-glycan different with glycopeptides of ionization unknown to due ing, processing is summarized in in summarized is processing N-glycan glycoprotein of analysis for site-specific The procedure the procedureof Overview • • • are selectively captured using treatment with periodate followed followed periodate with treatment using captured selectively are • • fragmentation for ETD and CID of a combination using when even a challenge, is still N-glycans multiple with a of all forms tion of glycopeptide identifica trypsin, and chymotrypsin proteases, two only using lowing key stages. key lowing ( mannose/hybrid- glycans (+3Da) (+203Da)or complex-type that are notoccupied (+0Da)or thatare occupied by high- to thepredicted sequence, aminoacid forglycosites peptide treatment isemployed to create unique masssignatures relative In thethird stage (Steps 26–36), sequential endoglycosidase thesubsequent during PNGaseFtreatment. peptides mini of from theIP2software package (Steps 38–53). RawConverterwith and processed usingmultiple components 41–53). MS1andMS2dataare The from extracted MSraw files Proteomics (IP2) software Pipeline package (stage 5, Steps for thethree glycosylation states isdone usingtheIntegrated Data processing peptides for identification and quantitation of (Step 37). resultingThe samplesare subjected to LC-MS/MS analysis heating toheating prevent of incorporation In thesecond stage (Steps 19–25), proteases are denatured by increaseglycosite theconfidence each detecting (Stepof 18). inorderand chymotrypsin, to maximize sequence coverage and digestion’,‘triple trypsin andacombination chymotrypsin, of several differentdigested with protease treatments, including retainingtion while protein sequence coverage. Proteins are at pH6(Steps 13–17)to deamida minimize non-enzymatic (Steps 1–12). Glycoproteins are then denatured andalkylated arethey dissolved inthebuffers thatcontain nonvolatile salts In thefirst stage, buffer exchangeglycoproteins for if isneeded Fig. 2 Other methods that use endoglycosidases to identify glycosites glycosites to identify use that endoglycosidases methods Other 27–29,6 a 4 ). . Exemplary, and perhaps the most relevant to this this to relevant most the perhaps and Exemplary, . 2 . The methods developed by the two groups focus focus groups two the by developed methods The . 27,2 8 . Quantitative measurements are also challeng 28,2 40,41,4 9 . Although the complexity is reduced by reduced is complexity the . Although 4 . However, typically, glycopeptides glycopeptides typically, However, . Figure 2 Figure 18 O-water into the Cter b and consists of the fol the of consists and 8,27–29, ------

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. ate detectable peptides that contain all the glycosylation sites glycosylation the all contain that peptides detectable ate gener to enough is chymotrypsin and digestion triple of nation combi a glycoproteins, most for whereas glycosites, few a only containing glycoproteins in glycosites all identify to able is alone syndrome coronavirus (MERS-CoV S-2P protein). Triple digestion respiratory East Middle of glycoprotein spike the and Envtrimer HIV the as such weights, molecular large with proteins cosylated gly heavily on glycosites all detecting for essential is digestions REAGENTS M peptides in glycosites of the released identification shift), indirect allowing (+1 acid is Da converted to mass aspartic asparagine glycosylated F, PNGase which in using released then are peptides The lectins. using enriched are or beads, hydrazide-activated to binding by containing asparagine to aspartic acid during ESI-MS analysis ESI-MS during acid to aspartic asparagine containing glycosites with peptides of the ratio by measuring be determined can sites glycosylation of occupancy site and directly, applied be digestion’) and combinations of Arg-C, trypsin, elastase and subtilisin (‘triple tion by chymotrypsin, a combination of trypsin and chymotrypsin, number different of protease digestions are used, including diges • • • • • dent identification dent for identification a given glycosite ( glycosite, confi for residues of sides highly on acid both allowing amino numbers of variable with peptides that contains of a series ladder sequence a produces it that is proteases, nonspecific and Another benefit of the use of multiple proteases, including trypsin complicatecan analysis data which Asp,to Asn of deamidation non-enzymatic the minimize denatured and alkylated at pH 6 instead of a mildly alkaline pH to proteases. of combination a with Proteolysis design Experimental occupancy. site of degree the to addition in processing, glycan of extent the about a second endoglycosidase,of Endo H, addition provides here, additional information described protocol the with comparison By Denaturation of proteases. of Denaturation the protocol (requires ~30 of specificity high in resulting needed, is purification column no ammonium bicarbonate and ammonium acetate, are used, so that Another key aspect of the protocol is that only volatile salts, such as retaining the ability to detect while the glycosites three of different identification glycosylation the states.and complexity reduced in result will trypsin as such proteases specific of use (e.g., plasma) cells, mixtures more-complex for However, glycosite. each for glycosylation of states three the of analysis semiquantitative and detection confident higher much for allows proteases multiple of For glycoproteinssingle or simple mixtures (e.g., viruses), the use in conversion of Asn to Asp upon removal of the carbohydrate, carbohydrate, the of removal upon Asp to Asn of conversion in resulting glycoproteins, of deglycosylation for employed is ment Ammonium acetate (CH Urea (MilliporeSigma, cat. no. U5128) Fisher Scientific) Water by theGenPure (purified Pro water system; purification Thermo Iodoacetamide (IAA; MilliporeSigma, cat. no. I1149) (Fisher BioReagents,DTT cat. no. BP172-5) ATER I ALS 40,4 6 6 1 (Step 18). Of note, a combination of all proteolytic proteolytic all of combination a note, Of 18). (Step . Alternatively, for a purified protein, PNGase F can can F PNGase protein, purified a for . Alternatively, 3 COONH µ g of starting material). g starting of 6 In the next stage, PNGase F treat F PNGase stage, next the In 5 . To sequence maximize coverage, a 4 ; MilliporeSigma, cat. no. A1542) © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. Supplementary TableSupplementary 1 Glycoproteins are Glycoproteins 2 1 ). 8 0 ------. .

total peptides. total during during the deglycosylation step instrument. Invertase produced by the yeast yeast MS the by same produced the Invertase by instrument. analyzed and replicates technical two least at controls. and Replicates teolysis can incorporate O incorporate can teolysis for pro used proteases and other trypsin active Residual 19–25). • • • • teins in these complex protein samples protein complex these in teins plex-type glycan (+3) plex-type glycan (+203), and glycan (+0), com high-mannose/hybrid-type no contain that glycosites for signatures mass different generate Endo H by F with followed PNGase is to employed glycopeptides treatment. endoglycosidase Sequential steps. deglycosylation the to prior °C 100 avoid To glycosites. to heating by protocol have the in used proteases all may denature we this, that peptides of identification O of Asn deamidation and/or the residues protocol, non-enzymatic of success overall the on check a As of treatment. endoglycosidase completion test to controls as used be can and respectively, glycans, and to complex-type be by occupied high-mannose-type evisiae LC-MS/MS analysis. LC-MS/MS ( glycopeptides intact of analysis for challenging is which modifications, multiple with peptides on sites glycosylation localize to us allows and peptides step. Removal of increases N-glycans the of efficiencies ionization to h that can 1 deamidation occur this during non-enzymatic minimize for conducted are reactions deglycosylation the so comple to tion, rapidly progress and efficient highly are digestions measurement of site-specific N-glycan processing of glycopro of processing N-glycan site-specific of measurement in accelerate the second, will and hydrophobicity dimension first the in charge the which on based in separated systematically are (MudPIT), peptides technology identification protein sional multidimen proteins, purified of glycosylation characterization site-specific for of sufficient is separation single-dimension Although glycoproteins. the of weights molecular large the and glycosylation heavy of a result as Elite Orbitrap by the generated results the in missed be may that sites those for information ing glycan-process site-specific provide to able are they Thus, Elite. Orbitrap an than sample given a for spectra MS/MS more times Orbitrap an as such Fusion or Lumos,Orbitrap speed, is more sensitive and scan generates several higher a with instrument An glycoproteins. most for coverage sequence satisfactory Elite, provides Orbitrap the A as 37). such (Step spectrometer, mass LC-MS/MS of high-resolution type any by analyzed be then can and a mass change of +3 when carried out in O in out carried when +3 of change mass a and no. A144-212) Hydrochloric (36.5–38%(vol/vol) acid HCl; Fisher Scientific, cat. Acetic (Fisher acid Scientific, cat. no. A38-212) Formic (MilliporeSigma, acid cat. no. F0507) effect oneffect human tissue, thepotential to organs, with damage respiratory mists. acidic andforms acid themistandsolution Both have acorrosive Ammonium bicarbonate(NH 18 -incorpation at the C terminus should be seen in <5% of the the of <5% in seen be should C terminus the at -incorpation and and α -1-acid glycoprotein from bovine serum are known are known serum bovine from glycoprotein -1-acid !

CAUT I ON In principle, the deglycosylated peptides peptides deglycosylated the principle, In Supplementary Fig. 1 Fig. Supplementary natureprotocols ( Concentrated hydrochloric isacorrosive acid Steps Steps 26–36, Ideally, each glycoprotein is digested in digested is Ideally,glycoprotein each 4 HCO 18 into the C termini of the peptides peptides the of termini C the into 6 7 3 ; MilliporeSigma, cat. no. A6141) , resulting in a high false-positive Fig. 2 6

| 8 Sequential treatment of of treatment Sequential VOL.13 NO.6VOL.13 . a ). The endoglycosidase ). Saccharomyces cer Saccharomyces protocol 18 -water (Steps (Steps -water | 2018

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1200 into aliquots and store the buffer at −20 °C for up to 2 months. in 10 ml water.of Adjust the buffer pH to 6 by adding acetic acid. Dispense Ammonium Ammonium acetate (100 mM, pH 6) REAGENT SETUP Software EQUIPMENT Acetonitrile (ACN; VWR, cat. no. 200004-350) 4- Ethylene bridged hybrid (BEH) 1.7-μm C18 resin (Waters, cat. no. 186003556) EDTA disodiumsaltdihydrate (MilliporeSigma, cat. no. E5134) Trypsin (Promega, cat. no. V5111) Arg-C (Promega, cat. no. V1881) glasses. and safety personal protectivewearing equipment, including labcoat, gloves, HEK 293-F cells as described in a previous study inaprevious HEK 293-Fcells asdescribed (HA, hemagglutinin virus and influenza laboratory-made, expressed in Calcium chloride dihydrate (CaCl Tris base(Fisher Scientific, cat. no. BP152-10) gloves.wearing modest toxicity while inahood doses handled insmall andshouldbe login.j IP2 (Integrated Proteomics Pipeline, v5.0.1; glyco_motif_filter, v1.0( Notepad++, v7.5.4( RawConverter, v1.1.0.19( Scientific) Fisher (Thermo system 1000 EASY the with equipped spectrometer Tribridmass Fusion Orbitrap an (ii) or Scientific) Fisher (Thermo system EASY-nLCII the with equipped spectrometer mass Trap-Orbitrap Ion Hybrid Elite Orbitrap an (i) either for optimized is below protocol the spectrometer: Mass Freezer to set −80°C(Forma Scientific) Kimtech Science Kimwipes tissues(Kimberly-Clark) Parafilm(VWR, cat. no. 52858-000) Fisher Vortex Genie 2(Fisher Scientific, cat. no. 12-812) Desiccator (Hach) Lyophilizer (Labconco) Microcentrifuge Cruz (Santa Biotechnology, cat. no. sc-358765) microcentrifuge 5415R(MilliporeSigma,Eppendorf cat. no. Z605212) NanoDrop 2000 spectrophotometer (Thermo FisherpH meter (MilliporeSigma, cat. no. Z283037) Scientific, cat. no. ND-2000) Water to bathset 30°C(Fisher Scientific, cat. no. 15-462-5Q) Incubator to set 100°C(Fisher Scientific, cat. no. 05-412-500) Incubator to set 56°C(Fisher Scientific, cat. no. 15-103-0514) Incubator to set 37°C(Fisher Scientific, cat. no. 15-103-0514) filter,Centrifugal 30kDa(MilliporeSigma, cat. no. MRCF0R030) filter,Centrifugal 10kDa(MilliporeSigma, cat. no. MRCPRT010) Low-binding pipette tips, 200 Low-binding pipette tips, 10 MTL-0150-BC) no. cat. (VWR, ml 1.5 tubes, microcentrifuge Biotix ResearchEppendorf Plus pipette (Eppendorf) made, expressed study inaprevious inHEK293-Fcells asdescribed PNGase F(New Biolabs, England cat. no. P0705S) Endo H(New Biolabs, England cat. no. P0702L) (Promega,Chymotrypsin cat. no. V1061) Subtilisin (MilliporeSigma, cat. no. P5380) Elastase (Promega, cat. no. V1891)  ( as described in a previous study inaprevious as described BG505 SOSIP.664 (laboratory-made, expressed trimer inHEK293-Fcells Glycoprotein interest: of In thisprotocol, we usethree example proteins: NaOH (MilliporeSigma, pellets cat. no. 221465) Transferrin (MilliporeSigma, cat. no. T8158) α IgM (MilliporeSigma, cat. no. I8260) IgG (MilliporeSigma, cat. no. I4506) Invertase (MilliporeSigma, cat. no. I0408) Fetuin (MilliporeSigma, cat. no. F3385) 18 eyes, skin, andintestines irreversibly. Handle while itinahood protocol 20–25 °C -1-Acid glycoprotein (AGP; MilliporeSigma, cat. no. G3643) O-water (97 atom% of O-water (97atom% of

µ CR m Jupiter C18(Phenomenex, cat. no. 04A-4396)

| I VOL.13 NO.6VOL.13 T s p I CAL ) ) for upto 1year. Store thereagent inadesiccator atroom temperature | https://notepad-pl 2018 http:// 18 http://fields O; MilliporeSigma, cat. no. 329878) |

µ natureprotocols µ l (Corning, cat. no. 4150) fields.scripps.edu/y l (Corning, cat. no. 4151) 1 0 ), MERS-CoV S-2Pprotein (laboratory- 2 ·2H

Dissolve 0.077 g ammonium acetateof .scripps.edu/rawconv 2 us-plus.org/download O; MilliporeSigma, cat. no. C3306) © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. http://goldfish.scr

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1 CAUT 0 ates/wp/?page_id=68 ). I ON ACN has / ) /v7.5.3.htm ipps.edu/ip2/

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l ) ) Chymotrypsin (0.5 Chymotrypsin intoDispense aliquots andstore thesolution at−80°Cfor upto 2months. F andredissolve itinanequal volume of Ammonium (100mM, bicarbonate pH8) water. Prepare fresh solution for each experiment. Iodoacetamide (500mM) solution for each experiment. PNGase F(glycerol-free) for upto 2months. Place itinto adesiccator andstore thebuffer atroom temperature Ammonium acetate (100mM, pH5.5) up to 2months. 1 mMHCl. into Dispense aliquots andstore thesolution at−80°C for 50 mMTris-HCl (pH7.8), 5mMCaCl Arg-C (0.5 volume water. to 500mlwith Store thebuffer at4°Cfor upto 2months. solution isadjusted NaOH. to ~8.0by of theaddition thefinal Bring EDTA disodiumsaltof The the notgo into solution thepHof will until stirrer.vigorously on amagnetic Adjust NaOH thepHto 8.0with pellets. Ammonium (100mM, bicarbonate prepared with to 1year. seal thealiquots, andstore them inadesiccator atroom temperature for up Elastase (0.5 −80 °Cfor upto 2months. provided by manufacturer. into Dispense aliquots andstore thesolution at 0.1% (vol/vol) Formic acidin ACN month. 1 to up for temperature room at solution the Store water. of DTT (500mM) DTT (pH 6). Prepare fresh solution for each experiment. Endo H activity assays Endo Hactivity for each experiment. Trypsin (0.5 and store thesolution at−80°Cfor upto 2months. that can be used. be can that column of type thus and limits pressure in differ only and nature in similar very are nLCII Easy and 1000 Easy systems. LC other for adjusted be to have may parameters (Thermo); pump nLCII Easy EDTA (1M, pH8) intoDispense aliquots andstore thebuffer at−20°Cfor upto 2months. CaCl water.with Store thebuffer at4°Cfor upto 2months. Adjust concentrated thepHto 7.8with HCl. thefinal Bring volume to 1liter 100 mMTris-HCl (pH7.8) and store thebuffer at−20°Cfor upto 2months. water.bicarbonate in10mlof Measure thebuffer pH. into Dispense aliquots LC setup LC EQUIPMENT SETUP ACN. Store thesolution atroom temperature for upto 1month. acid Formic (vol/vol) 0.1% solution atroom temperature for upto 1month. (vol/vol) ACN water. and950mlof acid. formic Add 1mlof Store the 0.1% (vol/vol) Formic acidin5%(vol/vol) ACN solution atroom temperature for upto 1month. (vol/vol) ACN water. and200mlof acid. formic Add 1mlof Store the 0.1% (vol/vol) Formic acidin80%(vol/vol) ACN SDS-PAGE to deglycosylation. used canbe determine completion of done before using thisprotocol. glycans on high-mannose shiftsof Gel concentrations according to themanufacturer’s shouldbe instructions thedesired glycoprotein Endoamount of H of aseries istreated with use. ammonium Dissolve bicarbonatein1mlof 0.0079gof ammonium bicarbonateinto adesiccator itbefore for atleast48hto dry 18 intoDispense aliquots andstore thebuffer at−20°Cfor upto 2months. water.acetate in10mlof Adjust thebuffer pHto 5.5by acetic adding acid. Subtilisin (0.5 into aliquots andstore thesolution at−80°Cfor upto 2months. Urea (8M) O-water (97 atom% of O-water (97atom% of 2 (1M)

The given information is for use with an Easy 1000 or or 1000 Easy an with use for is information given The

m

Dissolve 0.048 g of ureaDissolve in100 0.048gof m g/ m Dissolve 0.147 g of calcium chlorideDissolve dihydrate water. 0.147gof in1mlof g/ m g/ m

g/ m l) Dissolve 0.0077 g of DTT in100 DTT Dissolve 0.0077gof m Dissolve 10

l) m l) Add 186.12 g of EDTA·2HAdd 186.12gof m

l) Dissolve 20 Dissolve 0.5 mg of elastase in 1 ml of water. in1mlof elastase Dissolve 0.5mgof Dispense g/

Dissolve 0.5 mg of subtilisin in 1 ml of water. in1mlof subtilisin Dissolve 0.5mgof m A set of control inwhich adefined experiments of A set

18 l)

Lyophilize thesolution thatcontains PNGase O)

Dissolve 0.00925 g of iodoacetamideDissolve in100 0.00925gof

Dissolve 12.114 g of TrisDissolve 12.114gof water. in800mlof Dissolve 25 Add 1 ml of formic acid to 1 liter liter 1 to acid formic of ml 1 Add

µ Dispense the Dispense g of Arg-Cg of in20 µ g of trypsin intheresuspension buffer trypsin g of

Add 1 ml of formic acid to 1 liter of to acid formic Add 1liter of 1mlof 2

, 2mMEDTA. into Dispense aliquots Dissolve 0.077 g of ammoniumDissolve 0.077gof µ 18 g of chymotrypsin in50 chymotrypsin g of

O-water. Prepare fresh solution µ Dissolve 0.079 g of ammoniumDissolve 0.079gof 18 l of 100mMammonium acetate l of 2 O-water into 200- O to 300 ml of H O to 300mlof

µ µ

Mix 50 ml of 100% Mix 50mlof l of abuffer thatcontains l of l of water.l of Prepare fresh Mix 800 ml of 100% Mix 800mlof 18 O-water, pH8) 18

O-water. 2 µ

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l of l of

Place

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.  attached cap.  to minimizesignals derived from contaminants. higher sensitivity due tominimal protein loss. High-purity reagents should beusedfor samplepreparation and MS analysis sample preparation. Only volatilesaltsare usedinthe protocol and these canberemoved bylyophilization, resulting in 2| Buffer exchange for glycoproteins PROCE The columnisre-equilibrated with20 The columnisre-equilibrated with20 1| section (Step13). volume Sample injection Flow rate Mobile phases Column Gradient S Gradient S

tep tep

Gradient program forGradient program EasynLCII: forGradient program Easy1000:

CR CR Rinse the filterbyadding 100 Insert a10-kDacentrifugal filterinto atube. I I T T D I I URE CAL CAL Ifthe protein isalyophilized powder, then proceed tothe ‘Denaturation and alkylation of glycoprotein’ To minimizesampleloss, low-protein-binding microcentrifuge tubesand pipettetipsshould beusedduring 2- 3–5 200 nl per min formic acid in ACN B: 0.1% (vol/vol) formic acid in water A: 0.1% (vol/vol) capillary column eter × 25-cm length a 100- pressure-loaded into resin (Waters) BEH 1.7- µ g g sample µ E µ µ l l with a 1- to µ l of bufferAprior tothe injection of sample. l of bufferAprior tothe injection of sample. asy 1000 m inner diam T T µ ime (min) ime (min) m C18 140 135 130 125 105 240 210 200 150 20 10 0 0

© 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. µ ●

l of 100mMammonium acetate immediately before use. Sealthe tubewiththe -

T 2- 10 10 400 nl per min (vol/vol) ACN formic acid in 80% B: 0.1% (vol/vol) (vol/vol) ACN formic acid in 5% A: 0.1% (vol/vol) capillary column × 15 cm length 100- pressure-loaded into a (Phenomenex) 4- IMI µ µ µ g g sample m m Jupiter C18 l l with a 1- to µ N m m inner diameter E % % G asy n ~11h S S olvent B olvent B 100 100 100 100 60 10 40 25 0 0 0 0 5 LC

II

the Orbitrap Elite. the Orbitrap to use. Fusion below wereand settings detailed theOrbitrap The with used Mass spectrometer setup AGC, automatic gaincontrol. injection time MS1 maximum MS1 scan range MS2 resolution MS1 resolution voltage Electrospray mode Ionization P DDA method Charge states exclusion Dynamic Fragmentation Isolation MS2 AGC target injection time MS2 maximum threshold Intensity MS1 AGC target arameter

natureprotocols The instrument should be mass-calibrated prior mass-calibrated prior shouldbe instrument The O 3 3 s cycle time rbitrap fusion 2–7 2–7 selected CID CID at 35% quadrupole 2 2 400–1,500 Positive 5 5 × 10 4 4 × 10 250 250 ms m/z 2.5 2.5 kV 50 50 ms 120 120 k 15 15 k 5 5 5 s e with 4 4 5

| VOL.13 NO.6VOL.13 2 2 +1 +1 and unassigned m/z O protocol are rejected rbitrap elite CID CID at 35% 400–2,000 1,000 1,000 ms Positive with ion trap Top ten 5 5 × 10 1 × 10 200 200 ms 2.5 2.5 kV 120 120 k 15 15 k 60 60 s 500 | 2018

4 6 |

1201 © 2018 Nature America, Inc., part of Springer Nature. All rights reserved.  temperature for 1h. 13| 4|  11| 10|  lyophilization. after  17| ammonium bicarbonate at8,000gfor 50minat4°C.  molecular weight limitof 10kDa. 16| unstable. Perform the alkylation stepinthe dark, asiodoacetamide islight-sensitive.  14| alkaline buffers, ifpossible, inorder tokeep non-enzymatic deamidation toaminimum. Denaturation and alkylation of glycoprotein them withthe lyophilizer.  maximum recovery of proteins. 9| used instead of mildlyalkaline buffersduring bufferexchange.  glycoproteins (atleastthree times) with100mMammonium acetate(pH6). 5|  If the volume of the solution is<100 8| 6| 7|  3| 1202  12|  the dark atroom temperature for 45min. 15| DTT. adding protocol

PAUSE PAUSE CR CR CR CR CR CR CR CR CR CR Dissolvethe glycoprotein in100 RepeatSteps9and 10atleastfour times and combine the fractions. Collectthe solution and store itinalow-protein-binding microcentrifuge tube. Dispense the sampleinto fiveequal aliquots for the following proteolytic digestion. Buffer-exchange to100mMammonium bicarbonate (pH8)byusing acentrifugal filterwitha membrane nominal Add 2 Lyophilize500 Add 11

Wash the filter membrane with100 Repeat Steps6through 7at leasttwotimes. Centrifuge the device at8,000 Pipette 100 Centrifuge the device at8,000 Pipette the solution containing ~30 Centrifuge the device (the tubeand the filter)at8,000 | I I I I I I I I I I VOL.13 NO.6VOL.13 T T T T T T T T T T I I I I I I I I I I CAL CAL CAL CAL CAL CAL CAL CAL CAL CAL

PO PO µ µ

l of 500mMDTT (toafinal concentration of ~10mM)and incubate the solution at56°C for 1h. I I

STEP STEP STEP STEP STEP STEP STEP STEP NT NT STEP STEP l of freshly prepared 500mMiodoacetamide (toa final concentration of ~50mM)and incubate the solution in The samples can be stored at −80 °C or in liquid nitrogen for at least 2 weeks. The solution can be stored up to 1 week at −80 °C or in liquid nitrogen. | µ 2018 To completebufferexchange, itisimportant towashthe filteratleastthree times with100 To preserve activity of iodoacetamide, prepare iodoacetamide solution immediately before use, as itis The acidic buffer, 100mMammonium acetate, should beusedfor samplepreparation instead of mildly Ifvolatilesaltssuch asammonium acetateare added tothe buffer, itisimportant tocompletelyremove Wash the filter membrane withthe bufferatleastfivetimes afterbuffer exchange in order toachieve To minimizenon-enzymatic deamidation, the acidic buffer, 100mMammonium acetate(pH6),should be To completethe bufferexchange, itisimportant toextensively washthe filterthatcontains Protein concentration isestimated withthe NanoDrop A280assay. Do not dry glycoproteins after denaturation, as they may not be fully soluble in the buffer buffer the in soluble fully be not may they as denaturation, after glycoproteins dry not Do (1,000 down Spin l of 100mMammonium acetateinto the filter. µ l of the solution atroom temperature for atleast5h. |

natureprotocols © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. g g for 50minat4°C. for 50minat4°C. g µ µ , 25 °C, 1 min) all the solution from the walls of microcentrifuge tube before before tube microcentrifuge of walls the from solution the all min) 1 °C, 25 , l of 8Murea in100mMammonium acetate(pH6)and placethe solution atroom l, bring the final volume to100 µ µ l of 100mMammonium acetate(pH6). g of target protein into the filter. Sealthe tubewiththe attached cap. ●

T IMI N G ~7 h g for 50minat4°C. µ l withwater. µ

l of 100mM

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. 23| chymotrypsin (Step18D), and acombination of trypsinand chymotrypsin (Step18E)at100°Cfor 30s. 22| 21| 20| 19| Denaturation Denaturation of proteases ( (D) ( (B) ( P chymotrypsin (option D)oracombination of trypsinand chymotrypsin (option E). Aliquots A–Cwilllaterbecombined into a‘tripledigestion’ sample(Step21).The remaining twoaliquots are digested with combinations of proteases involving: Arg-C followed bytrypsin(option A),elastase(option B),and subtilisin(option C). 18| E C A rotease treatments (iii) (iii) (iii) (iii) (iii) (vi) (iv) ) ) (ii) (ii) (ii) (ii) (ii) ) (v) (i) (i) (i) (i) (i) Coolthe samples atroom temperature for 30s. Separately, heat the combined tripledigestion sample(Step21)and the samplesgenerated from digestion with Combine the peptide mixtures derived from Step 18A–C into a‘tripledigestion’ sample. Redissolveeachsamplein100 Lyophilizethe peptide mixtures derived from eachof the fiveprotease digestions (Step18A–E) for atleast5h. Fivealiquots (containing 6 A S E A C ubtilisin lastase combinationof trypsinandchymotrypsin

hymotrypsin rg- liquid nitrogen for atleast2weeks. for MS detection.   the ESIsignals of analytes.   EDTA tofinal concentrations of 5mMand 0.2mM, respectively. 1:20 (wt/wt).Bring the final volume to100 the single enzymes alone. resulting inincreased sequence coverage when combined withtrypsindigestion.  1:20 (wt/wt)and 1:13(wt/wt)respectively. Add chymotrypsin to the fourth aliquot of the denatured glycoprotein at a chymotrypsin/protein ratio of 1:13 (wt/wt). Incubate the reaction at37°Cfor 16h. Bring the final volume to500 Add trypsinand chymotrypsin tothe fifthaliquot of the denatured glycoprotein atenzyme/protein ratios of Incubate the reaction ina30°Cwaterbathfor 10h. Bring the final volume to500 Incubate the reaction at37°Cfor 4h. Bring the final volume to500 Add subtilisintothe third aliquot of the denatured glycoprotein atasubtilisin/protein ratio of 1:20(wt/wt). Incubate the reaction at37°Cfor 16h. Bring the final volume to500 Add elastasetothe second aliquot of the denatured glycoprotein atanelastase/protein ratio of 1:20(wt/wt). Incubate the reaction at37°Cfor 16h. Add sequencing-grade modified trypsintothe solution atatrypsin/protein ratio of 1:10(wt/wt). Redissolve the peptide mixture in500 Lyophilize the resulting peptide mixture for atleast3htoremove waterand volatilesalt. Incubate the solution at37°Cfor 4h. Add Arg-C toone aliquot thatcontains ~6 adequate digestion efficiency.  C

followed by trypsin

CR PAUSE CR CR CR CR I I I I I T T T T T I I I I I CAL CAL CAL CAL CAL

PO

I STEP STEP STEP STEP STEP NT ●

The peptide mixtures derived from combination proteolytic digestion can bestored at−80°Cor in T Donot incubate the reaction longer than4h,inorder toobtainappropriate lengths of peptides Donot add toomuch chymotrypsin tothe solution, asitcanbeself-digested, which willsuppress IMI The utilityof tripledigestion cangenerate higher sequence coverage thandigestion withany of Arg-C isabletocleaveatthe Cterminus of arginine residues, including sitesnext toproline, Sequencing-grade modified trypsinisabletodigest glycoproteins atpH6whilepreserving ● N

T G IMI µ ~24 h g of denatured glycoproteins each)are subjectedtotreatments withmultiple proteases and © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. N µ G l of water. µ µ µ µ ~10 h l with100mMammonium bicarbonate (pH8). l with100mMammonium bicarbonate (pH8). l with100mMammonium bicarbonate (pH8). l with100mMammonium bicarbonate (pH8). µ

l of 100mMammonium acetate(pH6).

µ g of denatured glycoproteins atanenzyme/protein ratio of µ l with100mMammonium bicarbonate (pH8).Add DTT and

natureprotocols

| VOL.13 NO.6VOL.13

protocol

| 2018

|

1203

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.  PNGase Ftreatment conducted in ‘MS1, MS2, and MS3’, inwhich bothMS1 and MS2 data are extracted from the MS raw files. 39| 38| 37|  25|  35| 34| 33| used todetermine completion of deglycosylation. amount of the desired glycoprotein withaseries of PNGaseFconcentrations. Gelshiftsof N-glycans onSDS-PAGE canbe  32| same volume of 30| proceed tocompletion.  28| experiments should bedone before using thisprotocol (seeReagent Setup).  which hasoptimal activityatpH5.5. remaining activeproteases willaccelerate incorporation of 31| Data Data analysis LC  S 24| 1204  36|  29|  27| 26| ? ment. protocol equential equential endoglycosidase treatment

TROU

-M PAUSE PAUSE PAUSE CR CR CR CR CR CR CR Add PNGase F to the Endo H–treated peptide mixtures at a minimum enzyme/glycoprotein ratio of 500 NEB units per 10 Dispense the reaction mixture into aliquots that contain ~2 Set‘Experiment Type’ as data dependent. Enable ‘Selectmonoisotopic m/zinDDA’. ‘Outputformats’ should beset to Add the MS raw filestothe ‘filestocovert’window ofRawConverter. Extract the MS1 and MS2 spectra from the MS raw filesusing the spectrum-converting software RawConverter. Lyophilizethe samplesfor atleast3htoremove any remaining volatilesaltsfrom the samples. Incubate the reaction at37°Cfor 1h. Sealthe microcentrifuge tubethatcontains the mixture. Lyophilizethe PNGaseFsolution for atleast1himmediately before useand redissolve the PNGaseFenzyme inthe Incubate the reaction at37°Cfor 1h. Add 20 RepeatSteps22and 23at least fivetimes. Setupthe LC-MS/MS system tocharacterize the deglycosylated peptides asdescribed inthe ‘Equipment Setup’section. Lyophilizethe Endo H–treated samplesfor atleast3himmediately before use. Inthe meantime, proceed toStep30. Add Endo Htothe peptide mixtures ataminimum enzyme/glycoprotein ratio of 250NEBunitsper10 Separately, redissolve eachof the three samplesin20

| S I I I I I I I VOL.13 NO.6VOL.13 T T T T T T T /M I I I I I I I B CAL CAL CAL CAL CAL CAL CAL LES

S PO PO PO analysis µ

H I I I STEP STEP STEP STEP STEP STEP STEP NT NT NT l of 100mMammonium bicarbonate (pH8)prepared with OOT ● 18 The peptide mixtures can be stored at −80 °C or in liquid nitrogen for at least 2 weeks. The samples can be stored in liquid nitrogen for at least 1 month. The Endo H–treated peptides can be stored up to 1 week at −80 °C or in liquid nitrogen.

| T O-water (seeReagent Setup). 2018 Eachglycoprotein isdigested intwoorthree technical replicates and analyzed bythe same MS instru The quantity of PNGaseFthatisneeded for deglycosylation canbedetermined bytreating adefined Steps31and 32should bedone asquickly aspossibletoreduce contact of the reaction mixture withair. Completeremoval of ammonium acetateinthe samplesensures thatthe following PNGaseFtreatment will The quantity of Endo Hneeded for deglycosylation may varyfrom protein toprotein. Asetof control Completeremoval of volatilesaltsfrom the samplesisimportant for the following Endo Htreatment, To completelydeactivate the proteases used, Steps22and 23should berepeated atleastfivetimes. Any I IMI N ● G N

| T

G IMI natureprotocols variable; hours to 1 d per glycoprotein N G 6–48 h per glycoprotein 18 © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. O-water. ●

T IMI N G ~7 h µ 18 l of 100mMammonium acetate(pH5.5). O-water into the Cterminiof peptides during the following µ g of peptides and store them in liquid nitrogen immediately. 18 O-water tothe PNGaseFsolution. µ g. - µ g. © 2018 Nature America, Inc., part of Springer Nature. All rights reserved.  charge states. 51|  ‘find missing peptide’: enabled, mass tolerance: 50| detected. Further verification is needed for these potential glycosylation sites. glycosylation siteswhen multiple spectra hitswith checked before they are removed. Asparagine residues thatare not locatedatthe motif should beconsidered aspotential ?  ≤ protein: per peptides of number minimum as: set are parameters The kit). software 48| formation from N-terminal glutamine residue (−17.026549Q)asvariable modifications. fixed modification. Set oxidation (+15.9994M), deamidation (+2.988261N),GlcNAc (+203.079373N), and pyroglutamate Orbitrap instrument). No enzyme specificityisconsidered for searching. Setcarboxyamidomethylation (+57.02146C)asa 47| 46| 45| 44| 43| peptide probabilities and false-discovery rates.  Add contaminant proteins: No. 42| IP2. of ‘database’ the with database, 41| target glycoproteins inFASTA format. 52|  and/or 49| 40| ? types. instrument other for be to determined need will threshold appropriate the and used, spectrometer mass and system LC of type the on dependent be will and α glycans) high-mannose by (occupied invertase as such glycoproteins model well-characterized using by done be should experiment control A instrument. to instrument from vary will and noise spectral from information to distinguish optimal as determined empirically was value study.This present the in column the onto loaded are glycoproteins is considered only when at least one of the three has a peak area of at least 5 × 10 × 5 least at of area peak a has three the of one least at when only considered is

0.05, and precursor delta mass cutoff: cutoff: mass delta precursor and 0.05, -1-acid glycoprotein (occupied by complex-type glycans) before setting the peak area threshold. Peak area values values area Peak threshold. area peak the setting before glycans) complex-type by (occupied glycoprotein -1-acid TROU TROU

CR CR CR CR CR Determine the abundance of eachpeptide from eachraw filebythe sum of the ion intensity peakarea overall identified Startalabel-free analysis using Census (another component of the IP2software package). The parameters are setas: Setthe mass tolerance at50p.p.m.for precursor ions and 20p.p.m.for fragment ions (MS2 spectra are detected inthe StartaProLuCID search inthe IP2(v5.0.1)software package. Uploadthe MS1 and MS2 filestoIP2. Uploadthe database toIP2. Uploadthe resulting filethatcontains the sequences of the target glycoproteins tothe ‘database’ window of IP2. Setthe parameters as:Source: Uniprot; Organism Name: BosTaurus; Generate reverse (decoy) sequences: yes; Use atext editorsuch asNotepad++ ( Combine the data derived from twoorthree technical replicates afteranalysis of eachMS runseparately. Filterthe results generated byDTASelect withthe software ‘Glyco_motif_filter’ to remove those peptides with Add the resulting file to a predefined database, such as the European Bioinformatic Institute Institute Bioinformatic European the as such database, predefined a to file resulting the Add Filter the results generated by the ProLuCID search by using DTASelect (v2.0, another component of the IP2 IP2 the of component another (v2.0, DTASelect using by search ProLuCID the by generated results the Filter I I I I I T T T T T I I I I B B N I CAL CAL CAL CAL CAL +203 modifications thatare not locatedatthe motif (N-X-S/T, Xcanbeany amino acid residue except proline). LES LES

H H

STEP STEP STEP STEP STEP OOT OOT Ion injection time isused tofurther normalize the resulting peakarea. Allpeptides with Sequence coverage of target proteins should be>95%. Reverse(decoy) sequences should begenerated and included inthe final database inorder toestimate I I To improve the accuracy of the method, a set of peptides with with peptides of set a method, the of accuracy the Toimprove N N G G © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. N +203 modifications thatare not locatedatthe consensus motif should be manually ≤ https://not 10 p.p.m. 10 ≤ 10 p.p.m.,retention time tolerance: N +203 modifications that do not contain the motif are consistently epad-plus-plus.org / ) toprepare afilecontaining the sequences of the N natureprotocols +0, +0, ≤ 8 ≥ . Of note, ~2 ~2 note, Of . 0.1 min. 2, spectrum false-positive rate: rate: false-positive spectrum 2, N +3, and and +3, N Bos Taurus Bos +203 modifications modifications +203 µ

| m of purified purified of m VOL.13 NO.6VOL.13 protocol protein protein | N 2018

+3

|

1205

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. techniques peptides withmultiple glycosites byMS/MS isstillchallenging, evenwithcombination of different typesof fragmentation combination proteolytic digestion followed bysequential endoglycosidase treatment. ● tides differwithvariable glycoforms higher sensitivity of the protocol (only 30 glycan, or complex-type glycan. This strategy converts the glycoproteomics analysis to a proteomics analysis, resulting in (Endo H followed by PNGase F) to create unique mass signatures for glycosites that have no N-glycan, high-mannose-type One of the distinguishing features of this protocol is the use of sequential treatment of glycopeptides with endoglycosidases Validation of sequentialendoglycosidase treatment thus milligram quantities of materials are generally usedfor typical glycoproteomics methods analysis. Glycosylatedpeptides, however, havemuch lowerionization efficiency during MS analysis relative topeptides, and glycoproteins are digested with specific proteases such as trypsin, resulting in (glyco)peptides that are suitable for LC-MS/MS This protocol isusedtodetermine site-specificN-glycanprocessing of glycoproteins. Inthe most widely usedstrategy, ANT Steps 37–52, data analysis: variable; hours to 1 d per glycoprotein, depending on complexity Step 36, LC-MS/MS analysis: 6–48 h per glycoprotein Steps 26–35, sequential endoglycosidase treatment: ~7 h Steps 19–25, denaturation of proteases: ~10 h Step 18, protease treatments: ~24 h Steps 13–17, denaturation and alkylation of glycoprotein: ~7 h Steps 1–12, buffer exchange for glycoproteins: ~11 h T Troubleshooting advice canbefound in ? 1206 occupied by underprocessed oligomannose, and those on invertase produced by the yeast To this end, we applied the protocol to assess site-specific N-glycan processing of two well-characterized model glycoproteins, sequential endoglycosidase treatments proceed to completion, avoiding mis-assignment of N-glycan processing status. a 51 48 36 S protocol

TROU

tep b T le le IMI I

C | VOL.13 NO.6VOL.13 I 1 1 PATE N B | LES G

Troubleshooting table. 2 8 D H . Quantitative measurement of glycopeptides iscomplicated bythe fact thationization efficiencies of glycopep OOT RESULTS cannot be downloaded by Census is empty or The output file generated Low Low sequence coverage of No peptides detectable deamidation are detected belong to non-enzymatic Too many peaks that digestion combination proteolytic No peptides detected after target glycoproteins P | roblem 2018 I N G |

natureprotocols S. cerevisiae © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 3

8 . Thisprotocol describes analternative waytoovercome these problems bythe useof T able µ by by Census target glycoproteins are not recognized by RawConverter or the names of the The MS1 file was not extracted correctly exposed to air for too long (Steps 30–33) 18 P The microcentrifuge tube that contains for too long may not be active (Step 18) Proteases stored in the buffer at −80 °C (Step 16) out completely after buffer exchange Glycoproteins may not have been washed properly The LC-MS instrument is not performing g of sample is needed for a complete analysis). The key to success is that the and ossible reason O-water O-water is not completely sealed or was 1 . α -1-acid glycoprotein from bovine serum α -1-acid glycoprotein are fully processed complex-type glycosylation reaction mixture with air as quickly as possible to reduce contact of the place it into a desiccator. Perform Steps 30–33 Seal the microcentrifuge tube completely and the target glycoproteins not recognized by Census from the names of using RawConverter. Delete spaces that are Extract the MS1 spectra from raw files again Make fresh protease solution target glycoproteins after buffer exchange to maximize recovery of Wash the filter membrane at least five times to specifications well-calibrated and working properly according Make sure that the LC-MS instrument is S olution 7 0 . Glycosites on invertase are 29,6 1 . Characterization of glyco

- - © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. N processing of least one of the three had a peak area of at least 5 × 10 assess site-specific N-glycan processing for glycoproteins display similar ionization efficiencies during ESI-MS analysis, further indicating that the protocol is able to semiquantitatively (unoccupied), aspartic acid (PNGase F–treated), and N-acetylglucosamine-linked asparagine residues at the glycosylation site, 1.2, suggesting slightly increased sensitivity for peptides with the of glycotypes ( (>98% of purple bars, treatment only was also applied to release N-glycans on Kif_BG505, resultingkifunensine in peptides treatment was effective with and Kif_BG505homogeneous had high-mannose glycans ( treatment, and as expected, glycosites comprised >95% high-mannose-type glycosylation (green bars), indicating that the model protein (hereafter referred to as Kif_BG505). N-glycans on Kif_BG505 were first removed by sequential endoglycosidase HIV-1 Envtrimer BG505SOSIP.664, expressed inthe presence of kifunensine (high-mannose only),wasselected as a high-mannose glycans orcomplex-type glycans are detected equally during MS analysis. To testthisassumption, the Another major assumption isthatthe endoglycosidase-treated peptide glycositesthatare unoccupied oroccupied by Validation of M complete denaturation of proteases used ( 18 ( Saccharomyces cerevisiae Figure 3 experiments. Mean ± s.e.m. were plotted. N275 ( as entirely high-mannose-type glycosylation, and site occupancy was >90% for all glycosites except the sites N64 and well as the previous study per site ( and site occupancy was >98% for all five sites ( treatment reached completion. Fig. +3, and O-incorporation into the C termini of peptides were found among all spectra hits identified, which is attributed to the 3 b a Fig. 3a ). All N-glycosites on both glycoproteins were identified with multiple MS/MS spectra ranging from 54 to >10,000

| Proportion of total Proportion of total N Validation of sequential endoglycosidase treatment. ( +203 modifications was displayed only when at least one of the three had a peak area of at least >5 × 10 S 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 upplementary 0 α -1-acid glycoprotein. ( 23 36 71118165 118 111 97 64 23 , Fig. 4 b S ). By contrast, the five N-glycosites of detectionof glycotypes 49 111 97 64 . ( c ). Peptides with b Fig. 4 ) Color-coded bar graph of the site-specific N-glycan processing of invertase. ( T 1 ables 2 0 , a set of peptides with b ). The resulting two samples were then mixed at a molar ratio of 1:1 in order to assess MS detection 118 d ) Color-coded bar graph of the site-specific N-glycan processing of a and , © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 6 266 165 b adapted from ref. Glycosite N +3 and 6 7 5 6 8 9 512 398 384 369 356 275 266 3 ). Low percentages of spectra hits that contain non-enzymatic deamidation or S 275 upplementary N 356 Fig. 3c +203 modifications are both detectable for each glycosite, witha ratio of 1.0to 1 a 6 384 369 N 0 e:Hg-ans ope Noglycan Complex High-mannose Key: ) Scatter plot of the site-specific N-glycan processing of invertase produced by the yeast , Nature Publishing Group. +0, , α d 8 -1-acid glycoprotein were completely complex-type glycosylation, ). These results indicated that sequential endoglycosidase N . As expected, the 14 N-glycosites of invertase were identified 6 +3, and 1 T . ables 4 398 512 N and N +203 modifications was considered only when at +3 modification. Synthetic peptides that carry asparagine d c 5 ). As described in the procedure section, as 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0 45 414136 104 94 57 34 Fig. 4 45 414136 104 94 57 34 α c ) Scatter plot of the site-specific N-glycan -1-acid glycoprotein. A set of peptides with natureprotocols a ). On the other hand, PNGase F 8 . Data were obtained from six independent Glycosite

| VOL.13 NO.6VOL.13 N +3 modification protocol

| 2018

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1207 © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. glycans at the glycosites highlighted in yellow were assigned based on the proportion of spectra hits because peak area did not reacha themolar thresholdratio ofof 1:1.5 × Peptides10 that had potential glycosites but were not glycosylated were not included. The proportions of high-mannose and complex-type transferrin ( at least >5 × 10 ( for OSTwasfound tobepartially glycosylated, inagreement withprevious studies mannose-type glycosylation ( by complex-type structures (N171,N332,and N395),whereas the other twosites, N402and N563,were primarily high- which isthe major antibody produced inthe primary immune response, three siteswere shown tobecompletelyoccupied Data were obtained from nine independent experiments. Mean ± s.e.m. were plotted. Image adapted from ref. Figure 5 trimer Env HIV the of processing N-glycan site-specific of analysis for developed initially was protocol the Although E 1208 Figure 4 glycoproteins. ( it is applicable to analysis of site-specific N-glycan processing of recombinant glycoprotein therapeutics ( therapeutics glycoprotein recombinant of processing N-glycan site-specific of analysis to applicable is it serum glycoproteins ( glycoproteins serum be entirely complex-type glycosylation, consistent with previous studies ( determined ( due cells whole as sensitivity. such high its systems to complex more in processing glycoprotein of characterization in useful be to likely also b protocol a xamples of theprotocol ) Site-specific glycosylation of Kif_BG505 that was treated with PNGase F only. ( Proportion of total Site-specific N-glycanprocessing of recombinantly produced therapeutic glycoproteins, including IgG and IgM, was 0.2 0.4 0.6 0.8 1.0

0 | VOL.13 NO.6VOL.13

| | Validation of MS detection of glycotypes. ( Application of the protocol for characterization of site-specific N-glycan processing of recombinant glycoprotein therapeutics and serum lgG1 180 c b ) and fetuin ( a c Proportion of total Proportion of total Proportion of total 8 a 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 1.0 Fig. 5a . Data were obtained from at least six independent experiments. Mean ± s.e.m. were plotted. – 0 0 0 d ) Site-specific N-glycan processing of the recombinant glycoprotein therapeutics IgG ( Kif_BG505, H+PNGaseFonly Kif_BG505, Endo H + PNGase F Kif_BG505, EndoH+PNGase | 0.2 0.4 0.6 0.8 1.0 813137 133 88 81317161015 8h1724222625313239353336323846414842616865637 625 618 611 462 448 411 406 398 392 386 363 355 339 332 301 295 276 262 234 197 185h 185e 160 156 137 133 88 813137 133 88 Kif_BG505, 1:1mixture 2018 0 , b d ). A set of peptides with ). Human serumIgG, which contains IgG1-4 withIgG1 and IgG2 asthe major isotypes, wasfound to Fig. 5c Fig. lgG2 176 |

natureprotocols 156 156 Fig. 5 , b d 615 8h1724222625313239355 339 332 301 295 276 262 234 197 185h 160185e 160 0.2 0.4 0.6 0.8 1.0 ), and soluble or membrane-bound envelope glycoproteins from viruses ( viruses from glycoproteins envelope membrane-bound or soluble and ), 0 e:Hg-ans ope Noglycan Complex High-mannose Key: 8e15 9 3 6 7 9 0 3 3 355 339 332 301 295 276 262 234 197 185h 185e © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. b ). The glycositeN563thatisproximal tothe CH4domain and thus isapoorsubstrate 7 3 9 0 563 402 395 332 171 N a +0, ) Site-specific N-glycan processing of Kif_BG505 that was treated with Endo H followed by PNGase F. N +3, and Glycosite e:High-mannose Key: lgM N +203 modifications was displayed only when at least one of the three had a peak area of Glycosite c ) MS detection of peptides that contain Complex 363 363 Fig. 5 c 0.2 0.4 0.6 0.8 1.0 8 9 398 392 386 8 9 398 392 386 0 a ) 32,49,5 24,7 3 630 432 a Transferrin ) and IgM ( 1 . d 1 406 406 2 adapted from ref. 0 . Ofthe fiveN-glycositeson IgM, , Nature Publishing Group. 411 411 b ), as well as the serum glycoproteins 448 448 d N 462 462 0.2 0.4 0.6 0.8 1.0 +3 and 0 611 611 1 0 , Nature Publishing Group. N 1 625 618 1 625 618 916176 156 99 +203 modifications at Fig. Fig. Fig. 5a Fig. Fetuin 6 637 637 ). It is is It ). ,

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8 . © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. of spectra hits because peak area did not reach the threshold of 5 × 10 glycoprotein ( respiratory syndrome coronavirus (MERS-CoV S-2P protein, processing of recombinant envelope glycoproteins derived from viruses, including the prefusion-stabilized spike glycoprotein ectodomain of Middle East two subdomains (589–751), subdomains two and S2 (752–1,291), contained the (367–588), RBD glycosites the including protein, S occupiedthe of regions other whereas largely18–353), (residues byS1-NTD the complex-typein found glycans ( ( site N104 the for except occupied, fully were protein the on glycosites 23 all that revealed S-2P) (MERS ectodomain protein S-2P MERS-CoV prefusion-stabilized a of processing glycan epitopes plotted. transfer to glycoproteins in those patients in type I CDGs, an increase of mono-glycosylated transferrin was found due to defects of oligosaccharide assembly and in CDG patients in the 1990s congenital disorders of glycosylation (CDGs). In particular, serum transferrin was first used to diagnose abnormal glycosylation mer, each of which comprises two noncovalently associated subunits, S1 and S2 and S1 subunits, associated noncovalently mer,two ofcomprises which each mono per glycans N-linked ~25 with kDa) large(~600 a trimer is protein S-2P site. MERS-CoV glycosylation each at status Figure 6 of effective vaccines for those viruses those for vaccines ofeffective development confound thus and surveillance immune from protein underlying the protect to shield a as serve glycoproteins a complex-type structure. Of note, we did not observe that the proteases used had biases on specific glycotypes glycotypes specific on biases had used proteases the that observe not did we note, Of structure. complex-type a The glycan N1176, which is in the epitope for antibody G4 and was reported to mask antibody recognition line withthe previous studies serum transferrin revealed that the two glycosites of this protein were entirely complex-type glycosylation ( target of neutralizing antibodies, and thus are the focus of vaccine development ofvaccine focus the are thus and antibodies, ofneutralizing target 89% of siteoccupancy, glycosylation, withfulloccupancy of twosites(N99and N156)and partial glycosylation of the third glycosite (N176, a c b Abnormal glycosylation of serumglycoproteins isa common feature invarious human diseases, such ascancers and Membrane-bound envelope glycoproteins of various viruses, such as MERS-CoV S-2P protein and HIV Env trimer, Env theare HIV and protein S-2P MERS-CoV as such viruses, various of glycoproteins envelope Membrane-bound Proportion of total Proportion of total Proportion of total 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0 0 0 b

| , Application of the protocol for characterization of site-specific N-glycan processing of virus envelope glycoproteins. ( c 15,57,6 81317161015 9 3 6 7 9 0 3 3 5 6 8 9 9 0 1 4 6 1 1 2 637 625 618 611 462 448 411 406 398 392 386 363 355 339 332 301 295 276 262 234 197 185e 160 156 137 133 88 adapted from ref. 66 23 56 2 2 3 4 6 4 8 483 285 246 165 144 133 126 122 63 45 38 22 c ). The proportions of high-mannose and complex-type glycans at the glycosites highlighted in yellow were assigned based on the proportion BG505 SOSIP.664trimer Influenza virushemagglutinin MERS-CoV S-2Pprotein 104 9 , suggesting that vaccine design efforts would benefit greatly from understanding the N-glycan processing N-glycan the understanding from greatly benefit would efforts design vaccine that suggesting , 125 Fig. 5 1 5 6 2 236 222 166 155 0 , Nature Publishing Group. 7 2 73,7 d . Normal transferrin has two N-glycosylation sites, each of which is fully occupied ). 4 185h . Another serumglycoprotein, fetuin,wasalsofound tobeentirely complex-type © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 5 4 . Importantly, some neutralizing antibodies to those viruses have glycan-dependent glycan-dependent have viruses those to antibodies Importantly,neutralizing . some 7 5 . Analysis of site-specific N-glycan processing of commercially available human 4 410 244 a ), influenza virus hemagglutinin from H3N2 strain A/Victoria/361/2011 ( 8 592 487 8 . Data were obtained from at least six independent experiments. Mean ± s.e.m. were Glycosite Fig. 6 Fig. 1 1 7 8 870 785 774 719 619 a ). High-mannose glycans were predominantly predominantly were glycans High-mannose ). 6 55,6 9 . Characterization of site-specific N- site-specific of Characterization . 9 natureprotocols . N-linked glycans on those envelope envelope those on glycans N-linked . 1,176 ,1 ,2 1,241 1,225 1,213 6 9 , was found to have Key: a

| – VOL.13 NO.6VOL.13 c No glycan Complex High-mannose ) Site-specific N-glycan b Fig. 5 ), and HIV-1 envelope 1,256 73,7 protocol Fig. 6 c 4 ), in , whereas | ,7 1,288 1,277 2018

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. N-glycan processing, in which high-mannose structures are first trimmed from from Man trimmed first are structures high-mannose which in processing, N-glycan of pathway the matched SOSIP.664 BG505 of site glycosylation each at identified glycans complex-type and mannose confirming the glycan composition at this site described in previous structural studies structural previous in described site this at composition glycan the confirming structures, high-mannose of predominantly composed Env, was HIV to PG16 and PG9 bnAbs the of binding for critical is occupied predominantly by underprocessed oligomannose, consistent with previous studies previous with consistent oligomannose, underprocessed by predominantly occupied be to found were region, patch high-mannose the in N392, and N386, N339, N332, N295, glycosites, the particular, In glycans. complex-type and high-mannose of mixture a had sites other six and glycosylation, complex-type >75% were four high-mannose, >75% were 14 occupied, largely were that those Of N625. and N618, N197, N185e, sites the ( glycosite per hits spectra 2,000 to up with glycosites 28 all of identification in resulting SOSIP.664, BG505 trimer Env HIV benchmark the to protocol the applied Wealso HA glycoprotein. this on occupied 32% only was N144 site another and occupied not was N122 site the that observe to striking is It ( glycans complex-type and ofhigh-mannose mixture a by occupied were rest the and N483), and N63, N38, (N22, glycosylation complex-type fully were four N285), and N165, (N45, high-mannose >85% were three Victoria/361/2011, regard to jurisdictional claims in published maps and institutional affiliations. com/reprints/index.htm at online available is information permissions and Reprints 7. 6. 5. 4. 3. 2. 1. CO AUT A online version of the pape Note: Any Information Supplementary and Source Data files are available in the The data thatsupportthe findings of thisstudy are availablefrom the corresponding author upon reasonable request. Data availability ( 1210 L.C. L.C. and J.C.P. wrote the manuscript. purified Env proteins. J.S.M., D.R.B., J.R.Y., and J.C.P. supervised the project. MS analysis. L.C. and S.-K.R.P. analyzed the data. N.W. and M.P. expressed and prepared samples for MS analysis. J.K.D., L.C., C.M.D., and Y.M. performed the (D.R.B. and J.C.P.), R01AI127521 (J.S.M.), and P41 GM103533 (J.R.Y.). (NIH) grants R01AI113867 (J.C.P., J.R.Y. and W.R. Schief), UM1 AI100663 those fieldswho want togainhigh-level information aboutthe glycoproteins they investigate. simple complex-type structures if they were occupied by a mixture of high-mannose and complex-type glycans complex-type and high-mannose of mixture a by occupied were they if structures complex-type simple intact glycopeptide level glycopeptide intact on obtained protein same the of results the to compared as glycans, complex-type/hybrid define that monosaccharides glycosylation, whereas other sites were occupied by mixtures of processed high-mannose structures (Man structures high-mannose processed of mixtures by occupied were sites other whereas glycosylation, S protocol cknowle

M Further information onexperimental design isavailableinthe We believe thisprotocol willbeof wide interest tothe proteomics and glycomics fields, and will beusedby many outside upplementary Fig. 2 Fig. upplementary H PET Proteomics 291 and disease. and system. immune the and Glycosylation infectivity.viral of loss in results and gp120, ofdegradation lysosomal and folding affects gp120 HIV-1 of Asn260 at N-glycan conserved highly the of Deletion de Vries, R.P. Vries, de M.L.A. Leoz, de C.I.A. Balog, health of mechanisms cellular in Glycosylation J.D. Marth, & K. Ohtsubo, R.A. Dwek, & I.A. P.,Wilson, Cresswell,T., P.M.,Elliott, Rudd, J. Balzarini, & I. Braakman, Quandte,M., K.O.,Francois, L., Mathys, glycans. N-linked of functionsIntracellular M. Aebi, & A. Helenius, cancer progression. cancer state affects receptor-binding specificity. specificity. receptor-binding affects state (2001). (2011). OR

| VOL.13 NO.6VOL.13 I

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. , 855–867 (2006). 855–867 , L.C., L.C., J.R.Y., and J.C.P. designed the research. L.C. High-mannose glycans are elevated during breast during elevated are glycans High-mannose

authors declare no interests. authors competing |

natureprotocols ). Of the 12 glycosites on the recombinant influenza haemagglutinin (HA) of A/ of (HA) haemagglutinin influenza recombinant the on glycosites 12 the Of ). 9 , e101181 (2014). e101181 , 2 9 . Thus, the sites were predominantly occupied by Man by occupied predominantly were sites the Thus, . Science

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285 cleaved envelope trimers. envelope cleaved on gp41 of conformationprefusion the on epitope glycan-dependent trimers. Env HIV-1 intact on interface gp41-gp120 the at epitope dependent 13800–13805 (2010). 13800–13805 antigens.oligomannoseentirely almost glycoprotein. envelope type of expressing cells and affect antibody recognition. antibody affect and cells expressing of type Med. HIV.of neutralizationbroadens gp120 of patch high-mannose antibodies. HIV potent circulation. the in glycoproteins of survival the determining in acid sialic of role The Care Crit. Resp. J. Am. mice. in immunopathology to contributes hemagglutinin virus influenza Kong, L. L. Kong, M. Raska, L.K. Pritchard, K.J. Doores, G. Ashwell, & J. Hickman, I.H., Scheinberg, G., Gregoriadis, A.G., Morell, Blattner,C. D. Sok, F.Garces, J.P. Julien, E. Falkowska, L.W.Cao, the of shielding Glycan J.A. McCullers, & K.L. Boyd, K., Wanzeck, K. Khatri, HIV-1 envelope trimers. envelope HIV-1 and multiple surrounding glycans. glycans. base V3 surrounding gp120 multiple and HIV-1 the of recognition via binding CD4 modulates (2016). interactions. host and microheterogeneity glycan virus A influenza for basis structural reveal 131–147 (2010). 131–147 immunogenicity.and antigenicity glycoprotein envelope (2013). S ciences , 20860–20869 (2010). 20860–20869 ,

c 6 ). All 28 glycosites were >90% occupied, except except occupied, >90% were glycosites 28 All ). , 236ra63 (2014). 236ra63 , et al. et Immunity et al. et et al. et et al. et et al. et et al. et J. Biol. Chem. Biol. J. et al. et et al. et et al. et Promiscuous glycan site recognition by antibodies to the to antibodies by recognition site glycan Promiscuous et al. et Expression-system-dependent modulation of HIV-1 HIV-1 of modulation Expression-system-dependent R et al. et Global site-specific N-glycosylation analysis of HIV of analysis N-glycosylation site-specific Global Structural evolution of glycan recognition by a family of family a by recognition glycan of evolution Structural Glycosylation patterns of HIV-1 gp120 depend on the on depend gp120 HIV-1 of patterns Glycosylation Integrated omics and computational glycobiology glycobiology computational and omics Integrated eporting Structural delineation of a quaternary,cleavage- a of delineationStructural

Broadly neutralizing antibody PGT121 allosterically allosterically PGT121 antibody neutralizing Broadly Envelope glycans of immunodeficiency virions are immunodeficiency virions of glycans Envelope 40 9 Broadly neutralizing HIV antibodies define a define antibodies HIV neutralizing Broadly if they were 100% high-mannose high-mannose 100% were they if Structural constraints determine the glycosylation of glycosylation the determineconstraints Structural 9 , 669–680 (2014). 669–680 , to Man to

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29080–29087 (2005). 29080–29087 2695–2706 (2016). 2695–2706 e02428-16 (2017). e02428-16 1598–1610 (2014). 1598–1610 research.epidemiological and genetic in G immunoglobulin of analysis glycosylation high-throughput glycoforms that can bind to mannan-binding lectin. mannan-binding to bind can that glycoforms based solid phase extraction. phase solid based chromatography-interactionhydrophilic using N-glycans serum human glycoprotein. envelope HIV-1 trimeric a of sites glycan gp140. soluble and trimers glycoprotein envelope membrane-anchoredHIV-1 trimers. Env eleven on based productionglycoprotein 667–672 (2003). 667–672 glycoproteins. N-linked identify to spectrometry 1285–1298 (2012). 1285–1298 glycoproteins. on heterogeneity glycan site-specific of (2010). 6154–6162 activator.plasminogen TNK-tissue therapeutic: protein follow-on the in site active the and occupancy glycosylation the of Biol. N-glycosylation sites. N-glycosylation glycosylation. in alterations associated cancer of identification efficient and fast for N-Glycans serum of profiling chromatographicliquid performance large B-cell lymphoma subtypes. lymphoma B-cell large diffuse of proteomics surface cell in-depth for enrichment glycosylation (2003). 660–666 spectrometry.mass and labeling isotope stable chemistry,hydrazide using glycoproteins N-linked of quantification Electrophoresis glycomics. structural for (HILIC)chromatography liquid interaction Chem. MSn. by details structural Mining 1. sequencing.carbohydrate for Res. structures. identification of rapid for library N-glycan serum a relationships. structure-functionfor implications patterns: glycosylation engineered with series hemagglutinin H5N7 influenza an of characterization Glycosylation sites. glycosylation relevant historically added sequentially with hemagglutinins virus A influenza H3N2 engineered of analysis Glycosylation (1985). spectroscopy.1H-NMR resolution high by determined IgM murine on sites glycosylation five at structures Chem. Biol. J. motifs. glycosylation (Asn-X-cys) atypical and (Asn-X-Ser/Thr) typical to N-glycans of additiondifferential from result CD69 human of forms dimeric Aldredge, D., An, H.J., Tang, N., Waddell, K. & Lebrilla, C.B. Annotation of Annotation C.B. Lebrilla, & K. Waddell, N., Tang, H.J., An, Aldredge,D., glycomics. of field emerging the and spectrometry Mass J. Zaia, Kaji, H. Kaji, andIdentification R. Aebersold, & D.B. Martin, X.J., Li, H., Zhang, J.E. Huffman, W.S.Characterization Hancock, & Karger,B.L. S.L., Wu, H.T.,Jiang, strategies V.Congruent Reinhold, & A. Hanneman, S., Singh, D., Ashline, P.C. Pang, L.W.Cao, Deeb, S.J., Cox, J., Schmidt-Supprian, M. & Mann, M. N-linked M. Mann, & M. Schmidt-Supprian, J., Cox, S.J., Deeb, and isolation affinity Hydrophilic Yoshida,S. & M. Tajiri,Y.,Wada, Determination Packer,N.H. & F. P.H.,Altmann, Jensen, D., Kolarich, hydrophilic in advances Recent Wuhrer,M. & Deelder,A.M. Zauner,G., O Mittermayr,S., J., Bones, A.J. Behrens, E.P.Go, E.P.Go, J.F. Cipollo, & C.A. Haan, R.P.,de Vries, deY., An, L.M., Parsons, J.F. Cipollo, & L.M. Parsons, I., Alymova, J.A., McCullers,Y., An, J.N. Arnold, carbohydrate W.J.Major Grimes, P.H.& Atkinson, D.R., Anderson, atypical of validation and Identification H. Zhang, & S.S. Sun, K.P.Multiple Kearse, & D.M. Segal, R.K., W.,Ribaudo, Wu, Vance,B.A., glycoproteomics. for glycopeptides of spectrometry mass tandem multiple-stage MALDI sialyl-Lewis(x) oligosaccharide on the zona pellucida. pellucida. zona the on oligosaccharide sialyl-Lewis(x) 1761–1764 (2011). 1761–1764

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` Experimental design 1. Sample size Describe how sample size was determined. Not applicable 2. Data exclusions Describe any data exclusions. Not applicable 3. Replication Describe whether the experimental findings were Each glycoprotein is digested in at least two technical replicates and analyzed by reliably reproduced. the same MS 4. Randomization Describe how samples/organisms/participants were Not applicable allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to Not applicable group allocation during data collection and/or analysis. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

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