Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on NAnoLC-MS2-product Dependent-MS3 ANAlysis of Permethylated Glycans*
2017; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês
10.1074/mcp.tir117.000156
ISSN1535-9484
AutoresCheng-Te Hsiao, Po-Wei Wang, Hua-Chien Chang, Yen‐Ying Chen, Shuihua Wang, Yijuang Chern, Kay‐Hooi Khoo,
Tópico(s)Genomics and Phylogenetic Studies
ResumoThe intrinsic Nature of glycosylation, Namely nontemplate encoded, stepwise elongation and termiNAtion with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and maligNAnt transformation. We have been pursuing this line of aNAlytical strategy that homes in on identifying the termiNAl sulfo-, sialyl, and/or fucosylated glycotopes by comprehensive NAnoLC-MS2-product dependent MS3 aNAlysis of permethylated glycans, in conjunction with development of a data mining computatioNAl tool, GlyPick, to eNAble an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping ameNAble to even nonexperts. We demonstrate in this work that diagnostic MS2 ions can be relied on to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS3 level. Both MS2 and associated MS3 data can be acquired exhaustively and processed automatically by GlyPick. The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification. We report here the technical aspects, reproducibility and optimization of such an aNAlytical approach that uses the same acidic reverse phase C18 NAnoLC conditions fully compatible with proteomic aNAlysis to allow rapid hassle-free switching. We further show how this workflow is particularly effective when applied to larger, multiply sialylated and fucosylated N-glycans derived from mouse brain. The complexity of their termiNAl glycotopes including variants of fucosylated and disialylated type 1 and 2 chains would otherwise not be adequately delineated by any conventioNAl LC-MS/MS aNAlysis. The intrinsic Nature of glycosylation, Namely nontemplate encoded, stepwise elongation and termiNAtion with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and maligNAnt transformation. We have been pursuing this line of aNAlytical strategy that homes in on identifying the termiNAl sulfo-, sialyl, and/or fucosylated glycotopes by comprehensive NAnoLC-MS2-product dependent MS3 aNAlysis of permethylated glycans, in conjunction with development of a data mining computatioNAl tool, GlyPick, to eNAble an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping ameNAble to even nonexperts. We demonstrate in this work that diagnostic MS2 ions can be relied on to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS3 level. Both MS2 and associated MS3 data can be acquired exhaustively and processed automatically by GlyPick. The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification. We report here the technical aspects, reproducibility and optimization of such an aNAlytical approach that uses the same acidic reverse phase C18 NAnoLC conditions fully compatible with proteomic aNAlysis to allow rapid hassle-free switching. We further show how this workflow is particularly effective when applied to larger, multiply sialylated and fucosylated N-glycans derived from mouse brain. The complexity of their termiNAl glycotopes including variants of fucosylated and disialylated type 1 and 2 chains would otherwise not be adequately delineated by any conventioNAl LC-MS/MS aNAlysis. One of the most conspicuous hallmarks of protein glycosylation is its extreme structural heterogeneity driven by nontemplate encoded stepwise elongation and branching from a few invariant core structures (1.Moremen K.W. Tiemeyer M. NAirn A.V. Vertebrate protein glycosylation: diversity, synthesis and function.NAt. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1099) Google Scholar). TermiNAl or peripheral sialylation, fucosylation and/or sulfation then generate a plethora of termiNAl glyco-epitopes, or glycotopes, distributed over myriad carrier glycans and proteins. MS-based glycomics (2.Zaia J. Mass spectrometry and glycomics.OMICS. 2010; 14: 401-418Crossref PubMed Scopus (184) Google Scholar, 3.Wuhrer M. Glycomics using mass spectrometry.Glycoconj. J. 2013; 30: 11-22Crossref PubMed Scopus (120) Google Scholar), despite being highly sensitive in affording high precision mass measurement at high throughput, often fails to reveal the least abundant glycotopes. A glycotope can be a very minor constituent of the glycome and yet be highly relevant when carried and presented properly at specific sites of receptors. Any change in abundance or additioNAl modifications imparted on this glycotope may well be undetectable at the glycomic level and yet will have profound impact on the functions of its carriers. This calls into question if current glycomics is of sufficient aNAlytical depth to address the most relevant glycobiology issues. Indeed, there is a considerable gap among positive detection of a glycotope by monocloNAl antibody or lectin, and its identification by MS. Although the most notable pitfalls of probing by antibody are its poorly defined cross-reactivity and not informative of carrier glycans, that of MS is a need to further resolve the isomeric or isobaric constituents of a glycotope defined by a unique mass. There will always be practical limitations in resolving each of the structural and stereoisomeric glycans chromatographically, particularly as the glycan size gets bigger along with increasing permutation of isomeric arrangements. MS2 and often MS3, if not higher orders, are needed to define linkage and substituent positions (4.Ashline D.J. Lapadula A.J. Liu Y.H. Lin M. Grace M. Pramanik B. Reinhold V.N. Carbohydrate structural isomers aNAlyzed by sequential mass spectrometry.ANAl. Chem. 2007; 79: 3830-3842Crossref PubMed Scopus (129) Google Scholar, 5.Reinhold V. Zhang H. Hanneman A. Ashline D. Toward a platform for comprehensive glycan sequencing.Mol. Cell. Proteomics. 2013; 12: 866-873Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In that respect, the most reliable MS-based glycan sequencing is based not on aNAlyzing NAtive but permethylated glycans (5.Reinhold V. Zhang H. Hanneman A. Ashline D. Toward a platform for comprehensive glycan sequencing.Mol. Cell. Proteomics. 2013; 12: 866-873Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6.Jang-Lee J. North S.J. Sutton-Smith M. Goldberg D. Panico M. Morris H. Haslam S. Dell A. Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies.Methods Enzymol. 2006; 415: 59-86Crossref PubMed Scopus (134) Google Scholar). In fact, the advantages of permethylation extend beyond that. We have previously shown that by converting all hydroxyl groups including the carboxylic groups of sialic acids into O-methyl, permethylation allows simple enrichment and identification of a wealth of sulfated glycans occurring at low abundance that would otherwise not be detected (7.Yu S.Y. Wu S.W. Hsiao H.H. Khoo K.H. ENAbling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.Glycobiology. 2009; 19: 1136-1149Crossref PubMed Scopus (47) Google Scholar, 8.Khoo K.H. Yu S.Y. Mass spectrometric aNAlysis of sulfated N- and O-glycans.Methods Enzymol. 2010; 478: 3-26Crossref PubMed Scopus (32) Google Scholar). We further demonstrated that negative ion mode NAnoLC-MS/MS can be productively applied in acidic buffer commonly used in proteomics without compromising much the stability and detection sensitivity (9.Cheng C.W. Chou C.C. Hsieh H.W. Tu Z. Lin C.H. Nycholat C. Fukuda M. Khoo K.H. Efficient mapping of sulfated glycotopes by negative ion mode NAnoLC-MS/MS-based sulfoglycomic aNAlysis of permethylated glycans.ANAl. Chem. 2015; 87: 6380-6388Crossref PubMed Scopus (20) Google Scholar). Low mass MS2 diagnostic ions for locating the sulfate onto the common Galβ1–3/4GlcNAc unit, with and without additioNAl sialylation, were identified and we suggested that ambiguity in assignment can conceivably be addressed by additioNAl MS3 mode that will not suffer from low mass cutoff the way an ion trap based fragmentation would (9.Cheng C.W. Chou C.C. Hsieh H.W. Tu Z. Lin C.H. Nycholat C. Fukuda M. Khoo K.H. Efficient mapping of sulfated glycotopes by negative ion mode NAnoLC-MS/MS-based sulfoglycomic aNAlysis of permethylated glycans.ANAl. Chem. 2015; 87: 6380-6388Crossref PubMed Scopus (20) Google Scholar). The advent of new generations of Orbitrap series with increasing data acquisition speed and flexibility in combining multiple stages of higher energy collision dissociation (HCD) with ion trap collision induced dissociation (CID) 1The abbreviations used are: CID; collision-induced dissociation; AGC, automatic gain control; DDA, data dependent acquisition; FA, formic acid; GUI, graphic user interface; HCD, higher energy collision dissociation; LacNAc; N-acetyllactosamine; lacdiNAc; N,N′-diacetyllactosamine; LeA/X; Lewis A or X; LeY/B; Lewis Y or B; NCE, normalized collision energy; pd-MS3; product dependent MS3; PGC, poros graphitized carbon; XIC, extracted ion chromatogram. 1The abbreviations used are: CID; collision-induced dissociation; AGC, automatic gain control; DDA, data dependent acquisition; FA, formic acid; GUI, graphic user interface; HCD, higher energy collision dissociation; LacNAc; N-acetyllactosamine; lacdiNAc; N,N′-diacetyllactosamine; LeA/X; Lewis A or X; LeY/B; Lewis Y or B; NCE, normalized collision energy; pd-MS3; product dependent MS3; PGC, poros graphitized carbon; XIC, extracted ion chromatogram., invites innovative multimode MS2/MS3 data acquisition and processing methods that would best address the most relevant glycomic features. An important concept to be advanced here is that any structural feature that can be defined by diagnostic ions at MS2 and/or MS3 level can thus be identified and relatively quantified based on its MS2/MS3 ion intensity and the frequency it was produced in an automated LC-MS2/MS3 aNAlysis. We have experimented with a glycotope-centric glycomic workflow, which aims foremost to delineate the various isomeric glycotopes by well-established diagnostic ions. Using the same acidic solvent system for both positive and negative ion mode data acquisition allows for rapid switching within or in between runs to probe for occurrence of target (sulfo)-glycotopes and to derive quantification indices for rapid comparison across as many biological sources. This allows us to ask how these sialylated, fucosylated and/or sulfated glycotopes were affected on pathophysiological activation, and/or genetically or chemically manipulated in vivo and in vitro, in a way never possible. Aided by our own in-house developed data mining tool, tens of thousands of MS2/MS3 spectra can now be meaningfully interrogated by nonexperts and relevant glycotope information extracted in a fully automated fashion to make glycomics more commonplace. Both Colo205, a human colorectal adenocarcinoma cell line, and AGS, a human gastric adenocarcinoma cell line, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). Colo205 was cultured in 90% RPMI 1640 medium (Life Technology, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Scientific, Waltham, MA) and AGS was cultured in 90% Ham's F-12 nutrient mix (Life Technology) supplemented with 10% fetal bovine serum (Thermo Scientific). Fucosylated glycan standards, including Lewis A hexaose (GLY055), Lewis X hexaose (GLY051), H antigen pentaose type 1, (GLY033–1), H antigen pentaose type 2, (GLY033–2), Lewis B pentaose (GLY056), Lewis Y pentaose (GLY052), and Sialyl Lewis X Pentaose (GLY053), were purchased from Elicityl OligoTech, Crolles, France. Male C57BL/6J (8–12 weeks old) were bred and maintained in the animal core of the Institute of Biomedical Sciences at Academia Sinica following the protocol approved by the InstitutioNAl Animal Care and Utilization Committee of Academia Sinica. Brain striatum tissues were carefully removed, minced into small pieces, and transferred into a 2-ml grinder and homogenize on ice using a homogenization buffer (1 mm EGTA, 1 mm MgCl2, 10 nm okadaic acid, 100 μm, phenylmethylsulfonyl fluoride, 40 μm leupeptin, 25 mm Tris HCl buffer, pH8.0) containing the 1X cOmplete™ Protease Inhibitor (Roche, Switzerland), and 1× phosStop (Roche, Switzerland). The homogeNAte was first centrifuged at 500 × g for 10 min at 4 °C to remove debris. The superNAtant was collected and centrifuged at 50,000 × g for 1 h at 4 °C to collect membrane fractions existing in the pellets. Pellets were suspended with an ice-cold lysis buffer (0.2 mm EGTA, 0.2 mm MgCl2, 30 nm okadaic acid, 40 μm phenylmethylsulfonyl fluoride, 0.1 mm leupeptin, 0.2 mm sodium orthovaNAdate, and 20 mm HEPES, pH 8.0 plus 1X cOmplete™ Protease Inhibitor and 1X phosStop) and stored at −80 °C until used. Harvested 1 × 107 AGS, Colo205 cells and membrane fraction from one mouse striatum were extracted by lysis buffer containing 1% Triton X-100 and centrifuged to collect the superNAtant. The superNAtants were subjected to reduction by 10 mm DTT at 37 °C for 1 h, alkylation by 50 mm iodoacetamide at 37 °C for 1 h in the dark, and then precipitation by TCA to a fiNAl concentration of 10%. The remaining detergents were further removed by cold acetone precipitation and the recovered proteins were digested overnight by trypsin (250 μg for cells or 50 μg for extracts from 1 mouse striatum) in 50 mm ammonium bicarboNAte at 37 °C, followed by same amount of chymotrypsin in the same buffer at 37 °C for 6 h. N-glycans were released by 3 U of PNGase F treatment and O-glycans by alkaline β-elimiNAtion from the de-N-glycosylated glycopeptide, as described (7.Yu S.Y. Wu S.W. Hsiao H.H. Khoo K.H. ENAbling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.Glycobiology. 2009; 19: 1136-1149Crossref PubMed Scopus (47) Google Scholar). Both the released N- and O-glycans were permethylated by the sodium hydroxide/DMSO slurry methods (8.Khoo K.H. Yu S.Y. Mass spectrometric aNAlysis of sulfated N- and O-glycans.Methods Enzymol. 2010; 478: 3-26Crossref PubMed Scopus (32) Google Scholar) at 4 °C for 3 h and separated into nonsulfated, monosulfated, and the multiply sulfated glycans by loading the neutralized reaction mixtures onto a primed Oasis® Max solid phase extraction (SPE) cartridge (Waters, Milford, MA) (10.Cheng P.F. Snovida S. Ho M.Y. Cheng C.W. Wu A.M. Khoo K.H. Increasing the depth of mass spectrometry-based glycomic coverage by additioNAl dimensions of sulfoglycomics and target aNAlysis of permethylated glycans.ANAl. BioaNAl. Chem. 2013; 405: 6683-6695Crossref PubMed Scopus (22) Google Scholar), and eluted off by 95% acetonitrile, 1 mm ammonium acetate in 80% acetonitrile, and 100 mm ammonium acetate in 60% acetonitrile/20% methanol, respectively. Before MS aNAlysis, aliquots from all fractions were additioNAlly cleaned up by applying to ZipTip C18 in 0.1% formic acid and eluted by 75% acetonitrile/0.1% formic acid. The permethylated glycan standards and samples were dissolved in 10 μl of 10% acetonitrile in 0.1% formic acid. An UltiMateTM 3000 RSLC NAno system (ThermoFisher Scientific) was interfaced to an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (ThermoFisher Scientific) via a PicoView NAnosprayer (New Objective, Woburn, MA) for NAnoLC separation at 50 °C using a 25 cm x 75 μm C18 column (Acclaim PepMap® RSLC, ThermoFisher Scientific) at a constant flow rate of 500 nL/min. The solvent system used were 100% H2O with 0.1% formic acid (FA) for mobile phase A, and 100% ACN with 0.1% FA for mobile phase B. A 60 min linear gradient of 25 to 60% B for O-glycan, and 30 to 80% B for N-glycan and mono-sulfated O-glycan, was used for eluting the permethylated glycans. Another EASY-nLC™ 1200 system was interfaced to an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (ThermoFisher Scientific) via a NAnospray Flex™ Ion Sources (ThermoFisher Scientific) for NAnoLC separation at 50 °C using the same 25 cm x 75 μm C18 column (Acclaim PepMap® RSLC, ThermoFisher Scientific) at a constant flow rate of 300 nL/min. The solvent system used were 100% H2O with 0.1% FA for mobile phase A and 80% ACN with 0.1% FA for mobile phase B. A linear gradient of 40 to 95% B in 70 min was used for aNAlysis of the permethylated mono-sulfated O-glycan. The Orbitrap Fusion™ Tribrid™ mass spectrometer was operated in positive mode for aNAlyses of nonsulfated N-glycans (m/z mass range 800–2000, charge state 2 to 4) and O-glycans (m/z mass range 500–1700, charge state 1 to 3). Top speed mode was used for data dependent acquisition at 3 s duty cycle. Full-scan MS spectrum was acquired in the Orbitrap at 120,000 resolution with automatic gain control (AGC) target value of 4 × 105, followed by quadrupole isolation of precursors at 2 Th width for higher energy collisioNAl dissociation (HCD)-MS2 at 15% normalized collision energy (NCE) ± 5% stepped collision, with 10 s dyNAmic exclusion applied. HCD MS2 fragment ions were detected in the Orbitrap aNAlyzer at 30,000 resolution at an AGC target value of 5 × 104. Target HCD-MS2 ions detected at high resolution and accurate mass (HR/AM) within 10 ppm were selected automatically for product-dependent MS3 (pd-MS3) acquisition in the ion trap using CID at an AGC target value of 1 × 104 and 30% NCE, followed by ion trap detection. The MS system was operated in negative ion mode for aNAlyses of mono-sulfated O-glycans (m/z mass range 700–2000, charge state 1). The same Orbitrap resolution and AGC target value were applied for MS1 but a 5 s top speed mode was used instead for parallel HCD/CID MS2 data dependent acquisition (9.Cheng C.W. Chou C.C. Hsieh H.W. Tu Z. Lin C.H. Nycholat C. Fukuda M. Khoo K.H. Efficient mapping of sulfated glycotopes by negative ion mode NAnoLC-MS/MS-based sulfoglycomic aNAlysis of permethylated glycans.ANAl. Chem. 2015; 87: 6380-6388Crossref PubMed Scopus (20) Google Scholar). The AGC target value and NCE for CID MS2 in the ion trap was set at 1 × 104 and 40%, respectively. For HCD MS2 to be detected in the Orbitrap aNAlyzer at 30,000 resolution, the AGC target values was set at 5 × 104 and NCE at 50% with ± 10% stepped collision energy. For additioNAl pd-HCD MS3 data acquisition, the AGC target value and NCE were set at 1 × 104 and 45%, respectively. All data were processed by in-house developed software GlyPick v1.0 and Xcalibur software v2.2. GlyPick was developed using Microsoft Visual Studio 2013 professioNAl edition in conjunction with Thermo Raw API (Version 3.0 sp3) for data extraction from Thermo raw file. Its parameter settings can be fine-tuned by user inputs via a graphic user interface (GUI) according to the specific data set to be aNAlyzed, and output along with the result files. GlyPick starts from picking out only those MS2 spectra containing at least 1 or 2 user-specified glycan fragment MS2 ions and then tracks each of the selected MS2 scan back to its preceding MS1 scan and associated pd-MS3 scans. The m/z value of the monoisotopic precursor will then be determined from accurately mass measured MS1 scan and listed alongside that of the triggered precursor, its peak intensity, elution time and scan numbers. The generated output file in Excel format also tabulates as many of the user-defined MS2 ions detected above specified intensity threshold and within the mass accuracy tolerance, along with the sigNAl intensities of each. For pd-MS3 scans, predefined diagnostic MS3 ions, if detected, will be used to identify specific glycotope in addition to a listing of all detected MS3 ions and their sigNAl intensities. The total number of spectra in which each of the specified MS2 and MS3 ions diagnostic of particular glycosylation features or glycotopes was detected will be counted and their respective intensities summed for the purpose of relative quantification. Because multiple pd-MS3 events (normally ≥ 3) were specified, as many MS2 events were actually triggered for the same MS1 precursor to isolate the respective MS2 ion for each pd-MS3 without acquiring the additioNAl MS2 data. For quantification purpose based on spectral counting and summing of ion intensity, the MS1 and MS2 scans were each duplicated for every additioNAl pd-MS3 scans to compensate for the time spent on the same precursor. This extrapolation may end up giving extra quantification weighting for MS2 scans containing more MS2 ions targeted for pd-MS3 but deemed justifiable and give a better approximation for the glycotope-centric aNAlysis. GlyPick can also mass fit the m/z values of identified monoisotopic precursors to glycosyl compositions, with or without considering additioNAl user-defined adducts, modifications, maximum and minimum number of allowed glycosyl residues, and simple rules of permissible combiNAtion derived from biosynthetic constrains. Either a single round or iterative rounds of fitting can be executed. The latter involves initial fitting without considering the extra permutations generated by cation adducts and under-methylation. MS2 scans fitted will then be removed and the remaining be fitted again by considering the extras. All monoisotopic precursors thus assigned to the same glycosyl composition can be optioNAlly grouped and their inferred MS1 peak intensities summed to provide a quantitative measure of relative abundance. GlyPick version 1.0 containing all features described in this work is available on request for testing while new features are being added and performance continuously optimized. All glycotopes reported were based on detecting their respective MS2 and MS3 ions, as extracted out from the MS/MS data set by GlyPick. The critical glycosyl linkages for discrimiNAting the various fucosylated glycotopes and location of sulfates can thus be uNAmbiguously defined but the Gal/GlcNAc identification was inferred directly from Hex/HexNAc without further verification. The anomeric configuration was likewise based on presumptive glycobiology knowledge and not further defined. MS1 precursors fitted with glycosyl compositions by GlyPick were not meant to imply glycan structural identification. Only select glycans with their MS/MS spectra manually interpreted and shown annotated with cartoon drawings are considered as structurally assigned to be singled out for discussion. Their monosaccharide stereochemistry and anomeric configurations were similarly not further verified. Most of the current offline NAnospray MS/MS or online NAnoLC-MS/MS aNAlysis of permethylated glycans were conducted in positive ion mode doped with sodium to promote sodiated molecular ions (11.Ashline D. Singh S. Hanneman A. Reinhold V. Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn.ANAl. Chem. 2005; 77: 6250-6262Crossref PubMed Scopus (184) Google Scholar, 12.NAirn A.V. Aoki K. dela Rosa M. Porterfield M. Lim J.M. Kulik M. Pierce J.M. Wells L. Dalton S. Tiemeyer M. Moremen K.W. Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural aNAlysis.J. Biol. Chem. 2012; 287: 37835-37856Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 13.Ritamo I. RabiNA J. NAtunen S. Valmu L. NAnoscale reversed-phase liquid chromatography-mass spectrometry of permethylated N-glycans.ANAl. BioaNAl. Chem. 2013; 405: 2469-2480Crossref PubMed Scopus (21) Google Scholar), which would afford more complete sets of fragment ions. In contrast, under acidic conditions, the protoNAted molecular ions would mostly yield highly abundant nonreducing termiNAl oxonium ions via cleavage at HexNAc, concomitant with characteristic elimiNAtion of substituents at its C3 position (14.Dell A. F.A.B.-mass spectrometry of carbohydrates.Adv. Carbohydr. Chem. Biochem. 1987; 45: 19-72Crossref PubMed Scopus (324) Google Scholar, 15.Egge H. Peter-Katalinic J. Fast atom bombardment mass spectrometry for structural elucidation of glycoconjugates.Mass Spectrom. Rev. 1987; 6: 331-393Crossref Scopus (235) Google Scholar). The advantages offered are two folds: (1) fully compatible with conventioNAl proteomic data acquisition workflow to allow convenient scheduling, because there is no need to change solvent and NAnoLC set-up, with no unwarranted introduction of sodium into sophisticated high-end MS instrument system; (2) abundant MS2 product ion to be selected for MS3, with reliable, characteristic MS3 ions informative of the linkage. The programmed data acquisition mode is called product-dependent (pd)-MS3, which would automatically isolate any of the targeted MS2 ions for further stage of HCD/CID fragmentation. As demonstrated against a panel of authentic standards (supplemental Fig. S1), each of the fucosylated glycotopes can thus be uNAmbiguously defined based on which glycosyl residue is elimiNAted from the C3-position of the GlcNAc+. For the smaller O-glycans, the RP C18 NAnoLC-separation afforded at elevated temperature is reasoNAbly satisfactory. To take a simple case example, the O-glycan with a Fuc1Hex2HexNAc2-itol composition from AGS cells was found to comprise both simple core 2 and extended core 1 structures with Fuc on either Gal or GlcNAc giving rise to H or LeX, respectively. These were resolved into at least 4 distinct major peaks, with structures carrying H eluting earlier than LeX, and the branched core 2 earlier than extended core 1 (Fig. 1). AdditioNAl structural isomers carrying LeA would elute slightly later than the LeX counterparts as demonstrated with similarly prepared and aNAlyzed O-glycan sample from colo205 cells. This is further corroborated by the elution order of type 1 versus type 2 chain carried on a nonfucosylated extended core 1 structure. A label free quantification based on extracted ion chromatogram (XIC) of the resolved peaks for these smaller O-glycans including Tn, sialyl Tn and T is thus possible (supplemental Fig. S2). However, as the size increases, so are the possible structural isomers, concomitant with the increase in the number of nonfully resolved peaks for each XIC. Similar pattern also extends to sulfated O-glycans aNAlyzed in negative ion mode, as described previously (supplemental Fig. S2, S3) (9.Cheng C.W. Chou C.C. Hsieh H.W. Tu Z. Lin C.H. Nycholat C. Fukuda M. Khoo K.H. Efficient mapping of sulfated glycotopes by negative ion mode NAnoLC-MS/MS-based sulfoglycomic aNAlysis of permethylated glycans.ANAl. Chem. 2015; 87: 6380-6388Crossref PubMed Scopus (20) Google Scholar). For the N-glycans, each of the Man5–9GlcNAc2 can be clearly separated but not the individual isomers, whereas multiantenNAry complex type structures with various degrees of fucosylation and sialylation were not efficiently resolved (Fig. 2) (16.Zhou S. Hu Y. Mechref Y. High-temperature LC-MS/MS of permethylated glycans derived from glycoproteins.Electrophoresis. 2016; 37: 1506-1513Crossref PubMed Scopus (42) Google Scholar). Nevertheless, the termiNAl fucosylated epitopes as detected by MS2 could be similarly targeted for MS3 to define the occurrence of Lewis versus H glycotopes.Fig. 2RP C18 NAnoLC separation of permethylated high mannose type and biantenNAry complex type N-glycans. Overlay plots of extracted ion chromatograms for the high mannose structures indicated that the Man5–9GlcNAc2itol N-glycans were well resolved from one another but not for their individual isomeric constituents. Larger complex type N-glycans, with and without sulfates, produced more complicated and not well resolved ion chromatograms, the uNAmbiguous identification of individual chromatographic peaks based on MS2 of the protoNAted molecular ions alone is not feasible but glycotopes carried on each can still be efficiently determined by MS2-pd-MS3 aNAlysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Typically with our Orbitrap Fusion Tribrid instrument settings and acquisition parameters, a duty cycle of 3 s (Top Speed mode) applied to a sample of relatively high complexity could fit in an average total of 25 MS2+MS3 scans for a positive mode NAnoLC-HCD-MS2-pd-CID-MS3 aNAlysis, with the MS1 and HCD-MS2 scan being mass measured in the Orbitrap at 120 K and 30 K resolution, respectively. This amounts to an average total of > 20,000 MS2/MS3 spectra acquired within the effective elution range of glycans. In negative ion mode for the sulfated glycans, we opted instead for NAnoLC-parallel CID/HCD-MS2-pd-HCD-MS3 at a duty cycle of 5 s. Both CID and HCD MS2 were similarly mass detected in the Orbitrap for high resolution and mass accuracy but the HCD-MS3 spectra were acquired in the ion trap after fragmentation in the HCD cell to increas
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