ON-LINE SEPARATIONS COMBINED WITH MS FOR ANALYSIS

ON-LINE SEPARATIONS COMBINED WITH MS FOR ANALYSIS OF GLYCOSAMINOGLYCANS

Joseph Zaia*

Department of Biochemistry,Center for Biomedical Mass Spectrometry,Mass Spectrometry Resource,Boston University School of Medicine,Boston,MA

Received 30March 2007;received (revised)24March 2008;accepted 15July 2008

Published online 27October 2008in Wiley InterScience (https://www.wendangku.net/doc/1110017661.html,)DOI 10.1002/mas.20200

The glycosaminoglycan (GAG)family of polysaccharides includes the unsulfated hyaluronan and the sulfated heparin,heparan sulfate,keratan sulfate,and chondroitin/dermatan sulfate.GAGs are biosynthesized by a series of enzymes,the activities of which are controlled by complex factors.Animal cells alter their responses to different growth conditions by changing the structures of GAGs expressed on their cell surfaces and in extracellular matrices.Because this variation is a means whereby the functions of the limited number of protein gene products in animal genomes is elaborated,the phenotypic and functional assessment of GAG structures expressed spatially and temporally is an important goal in glycomics.On-line mass spectrometric separations are essential for successful determination of expression patterns for the GAG compound classes due to their inherent complexity and heterogeneity.Options include size exclusion,anion exchange,reversed phase,reversed phase ion pairing,hydrophilic interaction,and graphitized carbon chromatographic modes and capillary electrophoresis.This review summarizes the application of these approaches to on-line MS analysis of the GAG classes.#2008Wiley Periodicals,Inc.,Mass Spec Rev 28:254–272,2009

Keywords:glycosaminoglycan;heparin;heparan sulfate;keratan sulfate;chondroitin sulfate;dermatan sulfate;hyalur-onan;mass spectrometry;liquid chromatography;capillary electrophoresis

I.BIOLOGICAL SIGNIFICANCE OF GAGs

The glycosaminoglycan (GAG)family of linear polysaccharides,found in mast cell granules,on cell surfaces,in extracellular matrices and in basement membranes,was ?rst identi?ed over 100years ago from cartilage.GAGs play structural roles in con-nective tissue,tethering cell surfaces to protein and proteoglycan molecules through interactions with lectin domains (Yang et al.,1994).The high concentration of sulfated GAGs also provides swelling pressure that is necessary for visco-elasticity in con-nective tissue.In addition,cell surface GAGs interact with a large number of growth factor families,growth factor receptors,cytokines and chemokines (Bern?eld et al.,1999;Perrimon &

Bern?eld,2000).GAGs are believed to act as co-receptors for many growth factors and growth factor receptors,interacting with both partners,either speci?cally or non-speci?cally (Lander,1998;Herndon,Stipp,&Lander,1999).Although it is believed that speci?c patterns of sulfation on the GAG backbone mediate growth factor–receptor interactions,efforts to gain a thorough understanding of these events have been limited by dif?culties in obtaining sequences.GAGs are polydisperse with respect to chain length and sulfation pattern,rendering them analytically challenging.

Traditionally,a GAG oligosaccharide must be puri?ed to homogeneity before analysis using a combination of chemical and enzymatic degradation (Conrad,1998)and chromato-graphic,electrophoretic (Turnbull,Hopwood,&Gallagher,1999),or mass spectrometric (Ernst et al.,1998;Rhomberg et al.,1998a,b;Venkataraman et al.,1999)detection to obtain the https://www.wendangku.net/doc/1110017661.html,plete structural analysis entails puri?cation of suf?cient material to allow for multiple degradative steps.On-line separations coupled with mass spectrometry have the signi?cant advantage that they are sensitive and do not require a puri?ed sample.

II.OVERVIEW OF GAG STRUCTURE

All GAGs consist of repeating disaccharide structures.Mature GAG chains re?ect modi?cation events that occur shortly after initial chain polymerization (Esko &Selleck,2002;Bulow &Hobert,2006).Heparin,heparan sulfate (HS),keratan sulfate (KS),chondroitin sulfate (CS),and dermatan sulfate (DS)are elaborated by sulfation of hydroxyl groups.The heparin,HS,CS,and DS classes are elaborated by epimerization of some uronic acid residues at the C5position.The heparin and HS classes are acted upon by N -deacetylase,N-sulfotransferase activity.The disaccharide repeat structures of the four classes of GAGs found in mammalian systems are shown in Figure 1.

A.Hyaluronan

The simplest GAG from the chemical point of view is hyaluronan,a molecule synthesized at and extruded from the plasma membrane (Toole,2000),consisting of [GlcA b 3GlcNAc b 4]n with n >2,000in extracellular matrices.The disaccharides are not chemically modi?ed and the molecule serves as a tether between a variety of extracellular matrix molecules and cell surfaces by virtue of hyaluronan-speci?c lectin domains on cell surface receptor CD44and ECM proteins,glycoproteins and

Mass Spectrometry Reviews,2009,28,254–272#2008by Wiley Periodicals,Inc.

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Contract grant sponsor:NIH grants P41RR10888and R01HL74179.*Correspondence to:Joseph Zaia,Department of Biochemistry,Boston University School of Medicine,670Albany St.,Rm 509,Boston,MA 02118.E-mail:jzaia@https://www.wendangku.net/doc/1110017661.html,

proteoglycans.Oligosaccharides of hyaluronan have important biological activities and therefore molecular weight analysis of hyaluronan is an important consideration(Mahoney et al.,2001). Methods used most often for molecular weight analysis of hyaluronans include size exclusion chromatography(SEC)and gel electrophoresis as reviewed in(Kakehi,Kinoshita,& Yasueda,2003).Both MALDI(Sakai et al.,2007)and ESI-MS techniques are appropriate for determining the size of oligosac-charides derived from hyaluronan(Roboz et al.,2000;Mahoney et al.,2001).Hyaluronan oligosaccharides may be distinguished from isobaric heparosan oligosaccharides using tandem MS (Zhang et al.,2008).

B.Chondroitin/Dermatan Sulfate

Chondroitin sulfate(CS)consists of repeating units of[GlcA b3-GalNAc b4]and may be sulfated on the4-and/or6-postions of GalNAc.DS is a CS variant in which a substantial fraction of GlcA residues are epimerized to IdoA,and the IdoA may be sulfated at the2-position.CS/DS oligosaccharides are expressed in patterned domains with respect to the distribution of uronic acid epimers and with respect to sulfation patterns(Cheng et al., 1994).In addition,the capping regions of CS chains from cartilage aggrecan have been found to vary speci?cally with development and during the onset of osteoarthritis(Plaas et al., 1998;West et al.,1999).MS has the potential to vastly increase the information concerning spatial and temporal expression of CS/DS domains in biological tissue(Seidler et al.,2007).

C.Keratan Sulfate

Keratan sulfate(KS)is a sulfated polylactosamine chain,the disaccharide repeat of which,[Gal b4GlcNAc b3],is identical to that found in antenna extensions of N-and O-linked glycans (Funderburgh,2000).KS consists of approximately50dis-accharide residues(Stuhlsatz et al.,1981)and is linked to the protein either as an extension to an N-linked glycan(KS I, cornea),or through a Ser/Thr bound linker structure(KS II, skeletal tissue).Biosynthesis of KS chains occurs with simulta-neous polymerization and sulfation,forming a pattern of unsulfated,monosulfated(Gal-GlcNAc6S)and disulfated (Gal6S-GlcNAc6S)disaccharide units.The non-reducing ends of corneal KS chains are modi?ed with a variety of capping structures including sialic acids and a3Gal(Tai,Huckerby, &Nieduszynski,1996;Tai et al.,1997;Huckerby,Tai,& Nieduszynski,1998).The existence of biologically regulated sequence epitopes of KS has been demonstrated in antigenicity studies(Mehmet et al.,1986;Tang et al.,1986)and the structures of KS chains are also known to vary with age(Lauder et al., 1998).A number of important papers concerning MS of KS have appeared(Oguma et al.,2001a;Karlsson et al.,2005;Zhang et al.,2005a,b),and it is clear that on-line separations will play an important role for future structural studies.

D.Heparin and Heparan Sulfate

HS,like CS,is bound to proteoglycan core proteins through Ser/Thr residues via a xylosyl linker(Varki et al.,1999).HS and heparin chains are synthesized as[GlcA b4GlcNAc a4]and subsequently modi?ed by an N-deacetylase/N-sulfotranferase enzyme that removes GlcN acetyl groups and replaces them with sulfate groups.This enzyme produces domains of high N-sulfate content,interspersed with those containing high N-acetate content.The N-sulfated domains may be acted upon by glucuronic acid C5epimerase,resulting in the conversion of GlcA residues to IdoA and be subsequently sulfated at

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3O-and/or6O-positions of GlcN and/or the2O-postion of HexA. The mature chains thus display a pattern of N-acetylated domains with low degrees of O-sulfation and N-sulfated domains with high degrees of O-sulfation.Heparin is expressed exclusively in mast cells bound to the serglycin proteoglycan,the carbohydrate chains of which are highly sulfated,corresponding predom-inantly to[IdoA2S-GlcNS6S]n(Gallagher&Walker,1985; Kjellen&Lindahl,1991).HS expressed on cell surfaces and in basement membranes is more diverse than heparin in that it contains a greater percentage of N-acetylated domains.The structures of these HS chains vary among different core proteins, cell types and cellular environments.

III.STRUCTURAL ANALYSIS OF GAGs

Successful mass spectrometric analysis of GAGs depends on an extraction method that is compatible with the separations system and MS detection.Classical biochemical extraction procedures, summarized in(Iozzo,2001;Vynios,Karamanos,&Tsiganos, 2002;Didraga,Barroso,&Bischoff,2006;V olpi,2006), typically require further development to enable use of MS-based detection.In particular,levels of salts and contaminants such as other carbohydrate classes,nucleic acids or lipids that may not be a problem when using optical detection may cause un-acceptable background ion counts when using MS.LC/MS-compatible extraction methods for analysis of tissue GAGs have recently been demonstrated(Zhang et al.,2005a;Hitchcock et al.,2006).

A.Depolymerization of GAGs

Puri?ed GAGs are often too large in size and heterogeneous to permit direct mass spectral analysis and are usually chemically or enzymatically depolymerized.The extent of such digestion may be engineered to achieve complete depolymerization,in which disaccharides are produced.The analysis of disaccharide composition is typically done to characterize the overall chemical character of a given GAG preparation.Speci?c enzymes are available for depolymerization of each GAG class, and details regarding their sources and activities are given in (Ernst et al.,1995).Generally speaking,lyase enzymes act to cleave hexosaminic bonds and the newly liberated non-reducing ends contain a4,5-unsaturated(D-unsaturated)uronosyl residue. Oligosaccharides produced by lyase action are distinguished by the loss of water,relative to a fully saturated structure, that accompanies glycosidic cleavage.Chondroitin lyases and heparin lyases are speci?c,available commercially,and often used in depolymerization of GAGs.GAG hydrolase enzymes cleave hexosaminic bonds by addition of water to produce fully saturated uronic acid at the non-reducing chain termini. Keratanases and testicular hyaluronidase are commonly used GAG hydrolases.Nitrous acid is used to cleave N-sulfated hexosaminic bonds at pH1.5or free hexosamine at pH4(Conrad, 1998).This chemical cleavage is used for analysis of heparins but may be used for analysis of CS/DS after de-acetylation of GalNAc residues.Nitrous acid degradation products contain an anhydromannose residue at the reducing end.IV.MASS SPECTROMETRY OF GAGs

The topic of mass spectrometric analysis of the GAGs has been reviewed recently(Zaia,2004,2005;Chi,Amster,&Linhardt, 2005;Minamisawa&Hirabayashi,2006).Mass spectrometric ionization of carbohydrates,including GAGs,has been reviewed recently(Zaia,2006).The following is a summary of some general points germane to LC/MS methods.In summary,GAGs are acidic molecules that produce abundant negative ions.Their acidity also makes them signi?cantly more fragile than peptides or less acidic glycans.Acidic residues are most stable when ionized in deprotonated form or paired with a cation.Thus,GAG oligosaccharides may be analyzed directly using negative mode MS techniques or in the positive mode when paired with a cation. For on-line separations,ESI is the primary ionization method used.

A.FAB MS

The use of FAB in the analysis of GAG oligosaccharides has been reviewed(Zaia,2004,2006).FAB was used to develop fundamental mass spectrometric principles for analysis of the GAG class.Unfortunately,it imparts suf?cient internal energy to GAG ions to cause fragmentation of the sulfate groups.In addition,the FAB technique is signi?cantly less sensitive than either MALDI or ESI.For these and other reasons,it is no longer widely used for analysis of GAG oligosaccharides.

B.MALDI-TOF MS

The amount of energy imparted during the vacuum MALDI process suf?ces to fragment polysulfated oligosaccharides (Juhasz&Biemann,1994,1995).This problem may be circum-vented by pairing the sulfated oligosaccharides with basic proteins or peptides,resulting in the observation of complexes between peptide and oligosaccharide in the positive mode. This technique has been widely used for the determination of sulfated GAG oligosaccharide mass values(Venkataraman et al.,1999).The potential for the use of room temperature ionic liquids as MALDI matrices for analysis of uncomplexed polysulfated oligosaccharides has been explored recently (Laremore et al.,2006).MALDI MS may be used for analysis of chromatographic fractions containing GAG oligosaccharides in an off-line mode.

C.ESI-MS

The development of ESI enabled mass spectrometric analysis of GAGs without in-source fragmentation problems(Takagaki et al.,1992,1994;Chai et al.,1998;Kim et al.,1998).Provided that source desolvation conditions are carefully optimized using commercially available standards,polysulfated oligosaccharides may be analyzed in the negative mode with minimal fragmenta-tion to the sulfate groups occurring during the desolvation process(Chai et al.,1998;Kim et al.,1998;Desaire,Sirich,& Leary,2001;Zaia&Costello,2001;Zaia,McClellan,&Costello, 2001;Saad&Leary,2003;Naggar,Costello,&Zaia,2004;Saad &Leary,2004;Saad et al.,2005).As a?owing technique,ESI

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allows direct detection of chromatographic ef?uents.Chromato-graphic mobile phases must contain only volatile components to be compatible with ESI.Therefore,the chromatography system must either be optimized using only volatile solvent components or an on-line solvent desalting device.For GAGs,SEC,reversed phase ion pairing,normal phase,or graphitized carbon chroma-tography may be operated using mobile phases compatible with ESI.Capillary electrophoresis may also be used with on-line MS detection.These applications are reviewed in detail below.

Recently,the electron detachment dissociation(EDD)has been used to dissociate GAG oligosaccharides using Fourier transform MS(Wolff et al.,2007a,b;Chi et al.,2008).EDD is a technique whereby a beam of electrons detaches an electron from a negatively charged precursor ion in the FTMS cell,resulting in the formation of an odd-electron species that dissociates to form product ions(Budnik,Haselmann,&Zubarev,2001;Zubarev, 2003).It has been demonstrated that native glycoconjugate glycans dissociate to form structurally informative cross-ring cleavages in higher abundances than observed using conven-tional collision-induced dissociation(McFarland et al.,2005; Adamson&Hakansson,2007).High abundances of cross-ring cleavages were also observed for GAG oligosaccharides(Wolff et al.,2007a),and it is possible to distinguish uronic acid epimers based on diagnostic product ions(Wolff et al.,2007b).EDD has been applied to the analysis of the CS/DS chain of the bikunin proteoglycan(Chi et al.,2008).

V.LC/MS OF GAGs

Some of the chromatography modes used for on-line LC/MS of N-and O-glycans are also used for GAGs.Because in many cases work on N-and O-glycans predates that on GAGs,a brief discussion of general glycan applications is provided at the beginning of each section below.The intent is to summarize the principles necessary to understand the applications to GAG separations.It is beyond the scope of this discussion to present an exhaustive review for N-O-,and lipid-linked glycan LC/MS.

A.SEC–LC/MS of GAGs

Classical large format size exclusion chromatography(SEC)has long been used for separation of the oligosaccharide products of GAG depolymerization reactions,and recent work has described the use of high performance SEC for this purpose(Ziegler& Zaia,2006).High performance SEC columns are available in formats that operate below100m L/min?ow rate range,the ef?uent of which may be infused with or without a post-column splitter into the mass spectrometer source.

The?rst work showing on-line SEC–LC/MS of GAGs was accomplished using an Amersham/Pharmacia/GE Health Sciences Superdex peptide column with a mobile phase consisting of30%methanol,10mM HCl(Zaia&Costello, 2001).In subsequent work,the use of ammonium salts as a solvent modi?er has been found to be preferable.On-line SEC–LC/MS using a Superdex peptide column with10%acetonitrile, 50mM ammonium formate at40m L/min has been used to characterize CS/DS glycoform distributions from puri?ed proteoglycans from different sources(Hitchcock,Costello,& Zaia,2006).The same LC/MS system has been used for the characterization of normal and osteoarthritic cartilage samples (Hitchcock et al.,2006).The signi?cance of this work is that SEC–LC/MS is compatible with the tissue extraction procedure and that on-line tandem mass spectrometry determines the CS/DS glycoform distribution in the tissue.One limitation of on-line SEC–LC/MS is that the chromatographic resolution depends on the column volume when other factors are held constant.As a result,scaling down of the chromatography dimensions comes at the expense of resolution.

The use of an ion suppression device for reducing the concentration of ammonium ions for the purposes of separation of heparin oligosaccharides has been shown(Henriksen,Ring-borg,&Roepstorrf,2004).The ion suppressor serves to remove the ammonium cations from the SEC column mobile phase,thus improving the signal strength from the heparin oligosaccharides. Using this system,extracted ion chromatograms have been produced for heparin oligosaccharides up to degree of polymer-ization(dp)14.Such mixtures are extremely complex,and the separation system enables deconvolution of the heparin oligom-ers.Figure2shows SEC–LC/MS results obtained on Tinzaparin, the active substance in the anticoagulant drug Innohep1a low molecular weight heparin drug produced from partial heparin lyase depolymerization of intact heparin(Henriksen,Ringborg, &Roepstorrf,2004).The UV(A)and total ion chromatogram(B) traces show a partially resolved distribution ranging from dp2to >dp14.Traces(C–H)show extracted ion chromatograms for the ion corresponding to the most abundant composition of each oligomer size.The most abundant disaccharide repeat in heparin is(IdoA2S a4GlcNS6S)n and trace(C)shows dp4containing this repeat.Traces(D–H)show that the number of sulfate groups on heparin oligomers?3nà1,where n is the number of dis-accharide repeats.For example,(E)shows dp8,corresponding to 4disaccharide repeats and11sulfate groups.Such compositions are typical of heparins.Mass spectra were summed for the13 regions indicated in(A and B),and the results are shown in Figure3.Regions9–13correspond to decasaccharides through octadecasaccharides,respectively.SEC–LC/MS with an ion suppressor has also been applied to the analysis of the antithrombin binding characteristics of various heparin prepara-tions(Seyrek,Dubin,&Henriksen,2007).Although the SEC resolution is low,the combination with MS detection allows extremely useful characterization of low molecular weight heparin samples.

B.Strong Anion Exchange Chromatography

LC/MS of Glycans

GAG chains are often subjected to anion exchange-based separations for both preparative and analytical purposes. HPLC-based strong anion exchange methods for separation of GAG chains have been described(Pye et al.,1998;Vives, Goodger,&Pye,2001).Although on-line LC/MS analysis typically requires a gradient of sodium chloride that precludes direct detection using mass spectrometry,an ion suppression device may be used to remove salts before analytes enter the ion source(Simpson et al.,1990;Conboy&Henion,1992).Recently

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a sub-millimeter diameter strong anion exchange high pH anion exchange column was used on-line with a Na ?on cation exchange capillary desalting device to enable detection using an ESI ion trap mass spectrometer (Bruggink et al.,2005a,b).Results

showed baseline chromatographic resolution with MS detection for a series of fructans from dp3to dp13.It was also possible to analyze G M1-gangliosides from human urine samples.There is potential for use of such a desalting device for strong anion exchange LC/MS of GAGs and other acidic glycans.

C.Reversed Phase LC/MS of GAGs

Although native carbohydrates are not retained on reversed phase stationary phases,derivatization with a hydrophobic group improves their chromatographic properties.Reductive amination is a robust method for attaching a single amine-containing alkyl group to the reducing end of native oligosaccharides,and several different tags have been used for this purpose (Anumula,2000,2006).The tag increases reversed phase retention and adds a chromophore and/or ?uorophore to the analyte carbohydrates to improve optical detection.Reductive amination also improves mass spectrometric ionization responses (Harvey,2000).

Reductive amination with pyridyl amine (PA)serves to increase the hydrophobicity of N-and O-linked glycans to the point that they are retained using a reversed phase chromatog-raphy column (Yamamoto et al.,1989;Kuraya &Hase,1996).In principle,the most highly retained compound in such an analysis is the reductive amination reagent itself.As the size of the glycan portion increases,so does the hydrophilicity of the tagged molecule.A two-dimensional chromatography system featuring SEC and reversed phase separation has been used to map PA-labeled oligosaccharides from glycoproteins (Kuraya &Hase,1996;Yamamoto et al.,1989).This method has been applied to analysis of partial acid hydrolysis products of glycoprotein glycans (Makino et al.,1996).An additivity rule was applied for the correlation of two-dimensional chromato-graphic elution position with glycan chemical structure (Naka-gawa et al.,1995).An off-line RP HPLC-MALDI TOF MS method has been used to compare the expression of PA-labeled N-linked glycans in murine dermis and epidermis (Uematsu et al.,2005).The 2-aminobenzoic acid label has also been used for RP LC/MS of N-linked glycans (Chen &Flynn,2007).For further details on use of RP LC/MS for reductively aminated glycans see (Wuhrer,Deelder,&Hokke,2005).

The following example demonstrates an LC/MS method for quanti ?cation of GAG metabolites from urine or plasma after derivatization using 1-phenyl-3-methyl pyrazolone (PMP)(Ramsay,Meikle,&Hopwood,2003).Lyophilized samples of urine or plasma were derivatized with PMP,excess reagent was removed by chloroform extraction,and the aqueous layer bound to a C18cartridge.The cartridge was dried and washed with chloroform,dried again,and eluted with 50%acetonitrile.PMP-derivatized sulfated mono-and disaccharides were then analyzed by infusion using negative ion ESI tandem MS.This method has been used to quantify mono-and di-sulfated HexNAc and monosulfated HexNAc-HexA in plasma of mucopolysacchar-idosis patients (Ramsay,Meikle,&Hopwood,2003).The method has been used to monitor dose response in enzyme replacement therapy for mucopolysaccharidosis type VI using an animal model (Crawley et al.,2004).On-line reversed phase LC/MS has been used to pro ?le PMP-derivatized oligosacchar-ides from mucopolysaccharidosis type IIIA patient urine

(Mason

FIGURE 2.A :Photodiode array (PDA)chromatogram of tinzaparin

recorded at 231–233nm.B :ESI total ion chromatogram of tinzaparin.C –H :Extracted ion traces corresponding to abundant GAGs with different degree of polymerization (dp).Mass spectra were summed within the 13regions shown in (a –b).Compositions are given as (X ,Y ,Z )where X ?number of monosaccharide units,Y ?number of sulfate groups,and Z ?number of acetyl groups (Henriksen,Ringborg,&Roepstorrf,2004).?2004John Wiley and Sons,Limited.Reproduced with permission.

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G U R E 3.S u m m e d m a s s s p e c t r a f r o m d i f f e r e n t r e g i o n s i n t h e T I C c h r o m a t o g r a m s h o w n i n F i g u r e 2

(H e n r i k s e n ,R i n g b o r g ,&R o e p s t o r r f ,2004).T h e c o m p o n e n t s a r e c a t e g o r i z e d b y (X ,Y ,Z ),a s d e ?n e d i n F i g u r e 2l e g e n d .?2004J o h n W i l e y a n d S o n s ,L i m i t e d .R e p r o d u c e d w i t h p e r m i s s i o n .

LC/MS OF GAGs

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et al.,2006).In this example,urine GAGs were partially puri?ed using anion exchange chromatography followed by size fractionation.The GAG fractions were derivatized with PMP, extracted with chloroform and analyzed using on-line reversed phase LC/MS.GAG oligosaccharides including some hexasac-charides were detected.A series of di-to hexadecasaccharides were detected.The PMP label has also been used to characterize GAG disaccharide markers in organ tissue in a mouse mucopolysaccharidosis animal model(King et al.,2006).The use of ion pairing agents to facilitate reverse phase binding interactions is also reported for separation of sulfated GAG oligosaccharides,see below.

D.Reversed Phase Ion Pairing LC/MS of GAGs Carbohydrates do not interact with hydrophobic stationary phases because they interact favorably with water in the mobile phase.GAGs,with their acidic character,are amenable to use of ion pairing agents to increase the degree to which binding occurs to the reversed phase.As reviewed(Garcia,2005),retention of charged analytes using ion pairing may be viewed as partitioning of the uncharged ion-pairs onto the hydrophobic stationary phase.An alternative view is that the ion pairing agent coats the hydrophobic phase and retention of analytes occurs through an ion exchange mechanism.Although ion pairing agents improve chromatographic properties,they often interfere with mass spectrometric detection due to their propensity to produce strong signals,thereby suppressing those of the analytes.

The inclusion of quaternary ammonium salts in the mobile phase enables direct separation of GAG disaccharides using a reversed phase chromatography column(Lee&Tieckelmann, 1980).Such reversed phase ion pairing chromatography systems remain popular for disaccharide analysis,particularly when combined with?uorescence-based detection.The formation of ?uorescent derivatives through post-column addition of cyanoa-cetamide(Toyoda et al.,1991)have enabled GAG disaccharides from sub-microgram quantities of biological samples.This method has enabled progress in understanding of GAG expres-sion in model organisms including Caenorhabditis elegans and Drosophila(Toyoda,Kinoshita-Toyoda,&Selleck,2000; Toyoda et al.,2000).It has also been applied to heparan sulfate disaccharide analysis from human liver samples(V ongchan et al.,2005),among other biological systems.RPIP HPLC using tributylamine in the mobile phase has been shown to produce similar chromatographic resolution of a complex mixture of heparin oligosaccharides as observed using anion exchange.

Retention in RPIP HPLC is dependent on electrostatic interactions between the acidic GAG oligosaccharides and the amine amphiphile(El Rassi,1996).In order for RPIP to be useful, a mobile phase system must be found that produces adequate ion pairing and remains volatile enough to be compatible for on-line MS detection.A systematic study of the properties of di-,tri, and tetra alkyl ammonium ions for on-line LC/MS of GAG oligosaccharides identi?ed5mM dibutylamine as a promising ion pairing agent(Kuberan et al.,2002).In an acidic mobile phase,it has suf?cient cationic character to pair with GAG oligosaccharides and is volatile.A capillary HPLC LC/MS separation was shown for unsulfated heparosan from dp6to dp40. It was also used for analysis of synthetic heparin pentamers.

Other researchers prefer15mM tributylamine/50mM ammonium acetate as the ion pairing agent for on-line LC/MS of heparin-derived oligosaccharides(Thanawiroon et al.,2004).As shown in Figure4,these authors analyzed oligosaccharides from a30%heparin lyase depolymerization of heparin.The UV and total ion mass chromatograms are compared in(A).The UV trace allows absolute determination of the molar quantity of D-unsaturated disaccharides using232nm absorbance.The mass dimension of the data enables a series of oligosaccharide compositions to be identi?ed that contain either the reducing or the non-reducing end of the parent heparin chain.This is made possible by the fact that the reducing and the non-reducing ends of the parent chain have unique masses with respect to those deriving from the internal portion.Thus,a series of compositions may be identi?ed that correspond to a sequence.The ability to identify such structures from GAG preparations is clearly an enabling technology for the proteoglycan?eld.Summed mass spectra for dp2-dp14are shown in(B–E).Ions corresponding to dp2–dp6consisting of(HexA2S b/a GlcNS6S)n,where n?1–3are observed at the same m/z value,576,distinguished by charge state.Ions corresponding to dp8–14(E)were detected with a tributylamine adduct.This method has been applied to disaccharide analysis for pharmacokinetics of oral heparin dose in human subjects(Mousa et al.,2007).Similar chromatographic conditions were used to analyze CS oligosaccharides derived from the bikunin proteoglycan(Chi et al.,2008).

Direct mass spectrometric analysis of chromatographic ef?uents containing millimolar concentrations of amines is feasible for mass spectrometers dedicated solely to analysis of GAGs.To analyze other compounds,extensive cleaning of the MS source and optics is necessary to reduce the strength of signals produced from the amines to acceptable background levels.Because such extensive cleaning can take considerable time,laboratories wishing to analyze compound classes for which ion pairing is not necessary are posed with a challenge.It is best not to infuse ion pairing agents into an instrument for which compound classes not requiring such additives must be analyzed.

FIGURE4.A:RPIP-HPLC separation of heparin oligosaccharides obtained from controlled(30%)

heparinase depolymerization of bovine lung heparin.A total ion chromatogram using negative ESI-MS

detection(upper trace)with peaks numbered and a UV chromatogram at232nm(lower trace)with degree of

polymerization(dp)of peaks are shown.The inset shows the expanded view of both the total ion

chromatogram and the UV chromatograms of higher oligosaccharides assigned to dp16–dp28by peak

counting.Negative mode ESI mass spectra of the fully sulfated heparin oligosaccharides ranging in size

from disaccharide(dp2,B),hexasaccharide(dp6,C)to tetradecasaccharide(dp14,D).The full scan spectra

(upper panel)and narrow range spectra showing isotope distribution(lower panel)are presented for the

oligosaccharides of dp2–dp6(Thanawiroon et al.,2004).?2004The American Society for Biochemistry

and Molecular Biology,Inc.,Reproduced with permission.

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The chemical nature of different GAG preparations may necessitate optimization of separate RPIP mobile phase compo-sitions(Henriksen,Roepstorff,&Ringborg,2006).One RPIP method was developed using25mM tripropyl amine,30mM acetic acid with a water/methanol gradient to separate partially depolymerized heparin preparations containing in excess of 200components.A second method was developed using40mM butyl amine,40mM acetic acid with a water/methanol gradient to separate size-fractionated heparin oligomers.An on-line ion suppressor was used to remove the relatively high concentration of ion pairing agents prior to the MS source.A partially depolymerized heparin mixture produced extracted ion chro-matograms with peak widths of approximately1min.Peaks corresponding to dp12with16,17,and18sulfates produced distinct extracted ion peaks with retention times increasing with number of sulfate groups.Deconvoluted mass spectra showed the presence of dp10–dp30oligosaccharides.It therefore appears that the separation system is a useful means for pro?ling extremely complex,partially depolymerized heparin mixtures. The second method,using butlyamine,is useful for separating size fractionated heparin.Different HPLC gradient programs were optimized for dp4and dp6oligosaccharides.

E.Hydrophilic Interaction Chromatography

LC/MS of GAGs

Although normal phase chromatography is an older chromato-graphic technique than reversed phase,it is used far less often for biomolecular separations.As described in a recent review (Hemstro¨m&Irgum,2006),hydrophilic interaction chromatog-raphy(HILIC)is normal phase chromatography in which a polar stationary phase is used with a less polar mobile phase and in which water is used as the strongly eluting solvent(Alpert,1990). HILIC has been widely used for separation of carbohydrates (Churms,1996).It offers the ability to bind and separate both charged and uncharged carbohydrates using a gradient from high to low organic content.For LC/MS of GAGs,amine and amide stationary phases are the most widely used for HILIC separations. Solvent modi?ers are required,and MS-compatible ammonium salts are often used for this purpose.The uses of these two stationary phases for on-line LC/MS of GAGs are described below.

1.HILIC LC/MS of GAGs Using Amine

Stationary Phases

Amino propyl silica was the?rst stationary phased used for HILIC separation of carbohydrates.Because formation of a Schiff base between the primary amino groups and the glycan reducing end aldehyde is a concern,amine HILIC columns are often used for separation of reduced or reductively aminated carbohydrates.Many manufacturers offer amino propyl silica columns and non-silica packings are available.Such phases are effective for separation of D-unsaturated GAG disaccharides (Hjerpe,Antonopoulos,&Engfeldt,1979;Lee&Tieckelmann, 1979).Although the amine-bonded stationary phase may be considered to act as a weak ion exchanger,isocratic elution conditions were used for separation of the disaccharides.

A method employing a gradient of increasing concentration of NaH2PO4was developed for separation of D-unsaturated disaccharides(Yoshida et al.,1989)and used for separation of reductively aminated GAG oligosaccharides(Kinoshita& Sugahara,1999).Amine stationary phases have been used with gradients of acetonitrile/water without modi?er to separate mixtures of glycan alditols liberated from gastric mucins (Hanisch et al.,1993).Such conditions would be compatible with on-line MS detection.

The oligosaccharide pro?les of glycoprotein glycans may be resolved using an amine-type HPLC technology using a dextran ladder as a reference(Guile et al.,1996).When used with ?uorescent reductive amination with2-aminobenzamide,this system produces a sensitive and reproducible correlation of released glycan mixtures with glucose unit values from the dextran ladder.The chromatography system has been used to construct a database of O-linked glycan structures(Royle et al., 2002).An amine-based HPLC method has also been used to map N-glycans reductively aminated with2-anthranilic acid(Anu-mula&Dhume,1998).Oligosaccharides were separated based on the number of sialic acids.Fucose variants for each sialylated glycoform produced distinct chromatographic peaks.For further details on the use of amine-type normal phase chromatography in mapping of glycans,see(Wuhrer,Deelder,&Hokke,2005). Mono-sulfated N-glycans have been analyzed using amino-bonded HPLC–ESI-MS using a water/acetonitrile/ammonium hydrogen carbonate pH8.0solvent system(Thomsson,Karlsson, &Hansson,1999).The high pH of the solvent system facilitates analysis of the glycans using negative mode ESI.

The following examples demonstrate the usefulness of amino columns for separation of sulfated glycans in combination with a multiple reaction monitoring(MRM)method for quanti?cation of speci?c digestion products among different tissue samples.An MS-compatible amine HPLC method was developed for on-line negative ESI-LC/MS of KS oligosacchar-ides derived from keratanase II digestion(Oguma et al.,2001a). The mobile phase consisted of0.01M ammonium formate pH9.4/acetonitrile operated under isocratic conditions.Under these conditions,two disaccharides differing by a sulfate group: Gal b4GlcNAc(6S)and Gal6S b4GlcNAc(6S)were easily sepa-rated using amine HILIC chromatography,as shown in Figure6 (Oguma et al.,2001a).The source conditions were set so as to favor loss of SO3on the part of the disulfated disaccharide, forming an ion at m/z462that is isobaric with the[MàH]àion for the monosulfated disaccharide.The m/z precursor ion was selected and dissociated under relatively high energetic con-ditions whereby an abundant product ion at m/z98was observed. The LC–mass chromatograms in(a–d)show the abundances for the two disaccharides among four tissue samples.This example illustrates both the usefulness of HILIC for LC/MS of sulfated GAGs and MRM for quantifying expressed oligosaccharides or fragments thereof in different tissues.The same chromatography method was applied with LC/MRM MS to the analysis of the D-unsaturated disaccharides produced from lyase digestion of HS extracted from brain,liver and tumor samples(Oguma et al., 2001b).Both MS and tandem MS were acquired on-line to differentiate some of the isomeric D-unsaturated HS disaccharide structures.

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2.HILIC LC/MS of GAGs Using Amide

Stationary Phases

Because the amide group is less basic and reactive than the primary amine,amide stationary phases have advantages for separation of acidic glycans.Retention on amide type HILIC columns is less sensitive to eluant pH and Schiff base formation with native carbohydrates does not occur(Hemstro¨m&Irgum, 2006).At this writing,there are few commercial choices for amide-type HILIC columns,with the TSK-gel Amide-80(Tosoh Bioscience,Montgomeryville,PA)and Glycosep-N(Prozyme LLC,San Leandro,CA)used most frequently.

Amide HILIC HPLC was applied to separation of D-unsaturated GAG oligosaccharides for the purposes of dis-accharide analysis using an acetate buffer with isocratic elution (Akiyama et al.,1992).Partial lyase digestion of CS/DS GAGs produces a series of D-unsaturated oligosaccharide products differing by disaccharide units.Such products may be separated very effectively using amide HILIC columns using a gradient of decreasing organic content with an acetate modi?er(Saitoh et al., 1995).In this work,the GAG oligosaccharides were reductively aminated using PA(Kon et al.,1991)to enable?uorescence detection.The resulting chromatograms consist of a series of peaks corresponding to CS/DS dp2–dp22with baseline separation.Off-line ESI mass spectra were acquired directly on the fractions to determine the m/z values and compositions of the GAG oligo-saccharides.This off-line LC/MS method was used in a series of studies on enzymatic reconstruction of CS/DS and hyaluronan oligosaccharides using the transglycosylase activity of testicular hyaluronidase(Takagaki et al.,1999,2000a,b,2002;Takagaki& Ishido,2000).A similar method has been used to fractionate CS isomers of urinary trypsin inhibitor(Kakizaki et al.,2007).

Mobile phase conditions originally developed for off-line amide-based chromatographic glycan mapping(Guile et al., 1996)have been applied for on-line LC/MS(Mattu et al.,2000).

A gradient of decreasing acetonitrile concentration relative to water is used with an ammonium formate modi?er to separate glycans.Results were correlated relative to glucose unit values, and exoglycosidase treatments were used to sequence neutrophil gelatinase

B O-glycans.The system is also applicable to structural analysis of N-glycans(Royle et al.,2003).

Capillary amide-HILIC chromatography has recently been used for on-line LC/MS of underivatized N-glycans(Wuhrer et al.,2004).These authors packed5m m amide HILIC particles into a75m m?100mm fused silica column and used a mobile phase containing ammonium formate with a gradient of decreasing acetonitrile content relative to water.A dextran ladder was well separated from dp3–dp10.The elution times increased with N-glycan size and acidity.The total ion mass spectrum showed a combination of protonated and alkali-adducted ions.The chromatography allows on-line tandem MS of eluting glycans.Thus,normal phase LC/MS is a complement to use of reversed phase or graphitized carbon chromatography (GCC)LC/MS for analysis of permethylated glycans.This chro-matography system was used to analyze non-speci?c protease digestion products of glycoproteins(Wuhrer et al.,2005).The chemical properties of such glycopeptide products are dominated by the glycan portion of the molecules.As a result,separation of glycopeptide glycoforms was observed.

Capillary amide-HILIC LC/MS has been used to character-ize CS/DS oligosaccharides from connective tissue(Hitchcock, Costello,&Zaia,2008).The tissue was digested with pronase and the GAGs isolated using an MS-compatible workup procedure(Hitchcock et al.,2006).As shown in Figure5A,it was possible to detect dp2–dp12oligosaccharides with baseline resolution using an ion trap mass spectrometer in the negative mode.In(B),a mass spectrum summed over18–55min is shown,and the monosaccharide compositions of the GAG ions are indicated.Similar chromatographic conditions were used to determine antithrombin-binding heparin oligosaccharides using a hybrid ion trap-Orbitrap mass spectrometer(Naimy et al., 2008).These examples demonstrate the usefulness of amide HILIC for LC/MS of GAG classes.

F.Graphitized Carbon LC/MS of GAGs

Packed charcoal liquid chromatography columns have been used in preparative separation of oligosaccharides for decades,but do not have acceptable physical properties for HPLC(Koizumi, 1996).Graphitized carbon chromatography(GCC)columns were developed for HPLC separations of isomeric and closely related compounds and applied to separation of carbohydrate compounds,including mono-and disaccharides and cyclo-dextrins(Koizumi,Okada,&Fukuda,1991).Retention by GCC has been described to occur by an adsorption mechanism and planar molecules exhibit increased retention over non-planar ones.For oligosaccharides of the same repeating unit structure, retention increases with degree of polymerization.GCC exhibits greater hydrophobicity than octadecylsilyl reversed phase stationary phases.The structured graphite surface of the GCC material confers exceptional physical and chemical stability.As a result,the entire pH range can be used with a variety of solvents and temperatures.It is not necessary to use salts in the mobile phase,and thus GCC is well suited for interface with mass spectrometry(Davies et al.,1992).Structural isomers may be separated using GCC.To avoid splitting of anomers,oligosac-charides are often reduced or reductively aminated prior to GCC.

Neutral and sialylated N-glycans may be separated using the same GCC mobile phase system(Davies et al.,1992).Sialo forms elute later in the gradient,indicating that the presence of acidic groups in?uences GCC retention.GCC LC/MS has been used to probe glycoprotein glycan heterogeneity(Kawasaki et al., 1999,2000).Recently,native glycan separations using GCC in microbore(Itoh et al.,2002)and capillary(Kawasaki et al.,2003) scales with on-line MS detection have been reported.GCC has been packed into a microchip device of dimensions50mm?75m m width and50m m depth that incorporates a short trapping cartridge and an electrospray needle(Nin?onuevo et al.,2005). Such devices are very robust due to the fact that the?uidics connections are made robotically.These studies demonstrate the potential of GCC as an MS-compatible separation system that may be scaled down and adapted to meet the needs of glycomics. GCC has been used for LC/MS of permethylated oligosaccharide alditols under conditions whereby oligosaccharide isomers are separated routinely(Costello,Contado-Miller,&Cipollo,2007).

Sulfated N-linked glycans have been analyzed using GCC (Kawasaki et al.,2001).Monosulfated N-glycans have been

LC/MS OF GAGs&

Mass Spectrometry Reviews DOI10.1002/mas263

analyzed using GCC-negative ESI-MS using a water/acetonitrile mobile phase system with5mM ammonium formate pH9.3as a modi?er(Thomsson,Karlsson,&Hansson,1999;Karlsson et al., 2004).The high pH of the mobile phase facilitates detection using negative mode ESI.The peak shapes are narrower than observed for separation of the same compounds using an amino HILIC column.GCC LC/MS using the same conditions has also been applied to analysis of O-glycans from glycoproteins and salivary mucins(Schulz,Packer,&Karlsson,2002).A signi?cant fraction of the O-glycans detected were monosulfated and one low abundance ion was detected the m/z of which was consistent with two sulfate groups.

Native CS disaccharides were analyzed using GCC with UV detection using a water/acetonitrile gradient containing tri?uoro-acetic acid modi?er(Davies et al.,1992).The results showed baseline resolution of a and b anomers for the disaccharides.GCC negative mode ESI-LC/MS has been applied to the analysis of enzymatic digestion products of hyaluronan,KS,heparin,and HS(Karlsson et al.,2005).The GCC column(100mm?0.32mm)was eluted using a linear gradient from0–24% acetonitrile with20mM ammonium bicarbonate modi?er. Heparin/HS disaccharides were reduced prior to analysis to prevent chromatographic splitting of a and b anomers.As shown in Figure7A and B,the disaccharide alditols elute between26 and29min in the linear gradient with an order of elution of monosulfated

to 264Mass Spectrometry Reviews DOI10.1002/mas

other factors.As shown in Figure8,GCC LC/MS was used to separate oligosaccharide alditols produced from keratanase digestion of bovine cornea KS(Karlsson et al.,2005).A series of peaks corresponding to monosulfated disaccharide (20.34min),trisulfated dp4(32.46min),and pentasulfated dp6 (36.35min)were observed.These results indicate that GCC may be used for separation of polysulfated GAG oligosaccharides.

G.Capillary Electrophoresis–MS of GAGs

Capillary electrophoresis(CE)has great potential as a separation system for mass spectrometric analysis of biomolecules includ-ing proteins(Gennaro,Salas-Solano,&Ma,2006),glycans (Zam?r&Peter-Katalinic′,2004),and small molecule meta-bolites(Monton&Soga,2007).Advances in CE–MS of carbohydrates have been reviewed recently(Campa et al., 2006)and the present discussion will focus on applications to the GAG classes.

Di-and oligosaccharides derived from GAGs are readily amenable to gel electrophoretic(Calabro,Hascall,&Midura, 2000;Calabro et al.,2001;Karousou et al.,2004)and capillary electrophoretic(CE)separations(Mao,Thanawiroon,& Linhardt,2002;V olpi&Maccari,2006)due to their acidic, negatively charged nature.In normal polarity mode,samples are separated using uncoated fused silica capillaries and a basic buffer system in which analytes are carried toward the cathode by virtue of the electroosmotic?ow.The least acidic compounds elute?rst using this mode of electrophoresis.In reversed polarity mode,the electroosmotic?ow is minimized through the use of an acidic buffer system,and only analytes,such as GAGs,that retain anionic character are able to migrate toward the anode. Disaccharides produced by depolymerization of GAGs using lyase enzymes may be detected directly using the232absorbance of the D-unsaturated uronic acid residue.GAG disaccharides may be analyzed in normal polarity using a borate buffer at pH8–9 (al-Hakim&Linhardt,1991)or reversed polarity at pH3–4 (Pervin,al-Hakim,&Linhardt,1994).Substantially improved sensitivity of detection has been achieved through the use of reductive amination of the disaccharides to incorporate 2-aminoacridone(AMAC)as a laser-induced?uorophore (Lamari et al.,2002;Militsopoulou et al.,2002;Militsopoulou, Lamari,&Karamanos,2003).

Direct coupling of CE to MS under conditions appropriate for GAG disaccharide analysis was?rst accomplished using ammonium acetate buffer to investigate the forward and reverse polarities(Duteil et al.,1999).Forward polarity using20mM ammonium acetate pH9.2and reversed polarity using530mM ammonium acetate pH3.5were shown to work with both positive and negative mode ESI-MS detection of heparin disaccharides. Positive mode ESI-MS entailed use of a sheath liquid containing formic acid and negative mode ESI one containing triethylamine. Similar negative ESI-MS conditions have been used to separate hyaluronan oligosaccharides(Kuhn et al.,2003).In this

case, Mass Spectrometry Reviews DOI10.1002/mas265

polyacrylamide coated fused silica capillaries were used with forward polarity separation in 40mM ammonium acetate buffer,pH 9.0.The hyaluronan oligomers were all similar in their electrophoretic migration times,and the MS data serve to differentiate them based on mass.Reversed polarity CE using 30mM formic acid pH 3.2has been used for CE/MS with a sheath ?ow of 2-propanol,5mM formic acid pH 3.2(Ruiz-Calero et al.,2001).Negative mode ESI-MS of a mixture of heparin/HS disaccharides showed separation based on number of sulfate groups.

An on-line sheathless CE/MS interface has been used for the analysis of acidic glycan mixtures using uncoated fused silica capillaries and a buffer system containing 50mM ammonium acetate adjusted to pH 12.0with ammonia (Zam ?r &Peter-Katalinic

′,2001).On-line negative mode ESI mass spectra were acquired using a Q-TOF instrument.CS/DS oligosaccharides

were analyzed ?rst using CE with off-line ESI-MS (Zam ?r et al.,2002)and then using an on-line sheathless interface (Zam ?r et al.,2004).On-line tandem MS data were acquired for a dp18CS/DS oligosaccharide with 11sulfate groups,and the data used to assign the sulfation pattern.MS-compatible CE conditions have been optimized for separation of heparin oligosaccharides derived from a partial lyase digestion (Gunay &Linhardt,2003).Use of ammonium salts at pH 8.5shows baseline separation for heparin dp4–14in the normal polarity mode.The electrophoretic resolution was improved by the addition of 10mM triethylamine to the buffer system.

Frontal analysis capillary electrophoresis (FACE)with MS detection has been used to detect complexes between antithrombin and a synthetic heparin pentasaccharide

(Fermas

FIGURE 7.Separation and detection of heparan disaccharides (100ng each)using negative ion graphitized

carbon LC –MS.Panes (A –F )include base peak chromatogram and single ion chromatograms (SIC)of detected [M àH]àions.Structures assigned with an asterisk (*)indicate that these structures were detected in these SIC due to in-source fragmentation with loss of sulfate (S)as [M àH àS]àions (Karlsson et al.,2005).?2005Elsevier Limited.Reproduced with permission.

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et al.,2007).Using FACE,a mixture of the protein and oligosaccharide is subjected to continuous electrokinetic injec-tion in ammonium carbonate pH8.5.The unbound protein migrates fastest,followed by the protein-oligosaccharide com-plex.The unbound pentasaccharide migrates much more slowly than does the protein.The protein and protein-oligosaccharide complex were detected using positive ion ESI-MS.Unbound pentasaccharide was detected using the negative ion mode.

At this time,with relatively few examples published,the use of on-line CE MS for GAGs is still under development.

VI.SUMMARY

As reviewed here,there are several useful options for on-line separations with MS of the GAG compound classes.The universal nature of the SEC mechanism makes it a robust separations system for comparing the structures of GAG from sources where comparatively high sample quantities(>5m g)are available.SEC is very robust and may be used effectively to separate a series of GAG preparations with high reproducibility. Anion exchange chromatography is a natural choice for separation of acidic glycans such as GAGs.It use requires an on-line ion supressor,and the robustness of such devices for removing the high concentrations of salt required have not been demonstrated.Reversed phase chromatography is attractive because the chromatographic conditions are similar to those widely used for peptides.The extent to which reductively aminated glycans are retained using reversed phase chromatog-raphy diminishes with the size and polarity of the molecules.As a result,the retention of reductively aminated GAG oligosacchar-ides>dp4is uncertain.The inclusion of alkylamines in the mobile phase allows separation of GAGs and other acidic glycans using RPIP.On-line MS detection of GAGs using RPIP is widely used,either with or without an ion suppressor.Infusion of alkylamines is not recommended for instruments for which compounds not requiring this additive must be analyzed. HILIC separations work well for LC/MS of all glycan classes, including the GAGs.Amine-HILIC may be used for separation of reductively aminated oligosaccharides.Amide-HILIC has recently been applied to reduced format(75m m internal diameter)chromatography of glycan classes and has been applied to LC/MS of CS/DS and heparin oligosaccharides. GCC produces the highest resolution of any chromatography system for glycans including GAGs.It use for on-line LC/MS

has Mass Spectrometry Reviews DOI10.1002/mas267

been demonstrated from GAG disaccharides and poly-sulfated oligosaccharides.There appears to be potential for expanded use of GCC for LC/MS of GAGs.Although GAG digestion products are amenable to CE separations due to their negative charge,only a few reports have shown on-line MS analysis of GAGs with CE, and it appears that technical challenges remain.

VII.ABBREVIATIONS

CE capillary electrophoresis

CS chondroitin sulfate

dp degree of polymerization

DS dermatan sulfate

ESI electrospray ionization

FACE frontal analysis capillary electrophoresis

GAG glycosaminoglycan

GCC graphitized carbon chromatography

HILIC hydrophilic interaction chromatography

KS keratan sulfate

LC liquid chromatography

MALDI matrix-assisted laser desorption/ionization

MS mass spectrometry

PA pyridyl amine

RP reversed phase

RPIP reversed phase ion pairing

SEC size exclusion chromatography

ACKNOWLEDGMENTS

The author acknowledges support from NIH grants P41RR10888 and R01HL74197.

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Joseph Zaia received his Ph.D.in organic chemistry from MIT in1993,working in the

laboratory of Klaus Biemann on peptide sequencing by mass spectrometry.After a one year

post-doctoral fellowship at MIT,he did a second post-doc with Catherine Fenselau at the

University of Maryland,focusing on mass spectrometry of metalloproteins.He joined

the cartilage biochemistry group of Osiris Therapeutics,Inc.,in June,1996and worked on

the characterization of glycoproteins and proteoglycans from connective tissue.In June,

1999he became Assistant Research Professor of Biochemistry at Boston University School

of Medicine.Since this time he has served as Associate Director of the Mass Spectrometry

Resource,an NIH/NCRR-funded research resource center.He was promoted to Associate

Research Professor in2005.His research focuses on correlating structure and function for

heparan sulfates,and other glycosaminoglycans,using mass spectrometry as a primary tool.

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272Mass Spectrometry Reviews DOI10.1002/mas

with复合结构专项练习96126

with复合结构专项练习（二） 一请选择最佳答案 1）With nothing_______to burn，the fire became weak and finally died out. A.leaving B.left C.leave D.to leave 2）The girl sat there quite silent and still with her eyes_______on the wall. A.fixing B.fixed C.to be fixing D.to be fixed 3）I live in the house with its door_________to the south.（这里with结构作定语） A.facing B.faces C.faced D.being faced 4）They pretended to be working hard all night with their lights____. A.burn B.burnt C.burning D.to burn 二：用with复合结构完成下列句子 1）_____________（有很多工作要做），I couldn't go to see the doctor. 2）She sat__________（低着头）。 3）The day was bright_____.（微风吹拂） 4）_________________________，（心存梦想）he went to Hollywood. 三把下列句子中的划线部分改写成with复合结构。 1）Because our lessons were over，we went to play football. _____________________________. 2）The children came running towards us and held some flowers in their hands. _____________________________. 3）My mother is ill，so I won't be able to go on holiday. _____________________________. 4）An exam will be held tomorrow，so I couldn't go to the cinema tonight. _____________________________.

With的用法全解

With的用法全解 with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、 with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词； 2. with或without-名词/代词+副词； 3. with或without-名词/代词+介词短语； 4. with或without-名词/代词 +动词不定式； 5. with或without-名词/代词 +分词。 下面分别举例： 1、 She came into the room，with her nose red because of cold.（with+名词+形容词，作伴随状语）

2、 With the meal over ， we all went home.（with+名词+副词，作时间状语） 3、The master was walking up and down with the ruler under his arm。（with+名词+介词短语，作伴随状语。） The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.（with+名词+不定式，作伴随状语）He could not finish it without me to help him.（without+代词 +不定式，作条件状语） 5、She fell asleep with the light burning.（with+名词+现在分词，作伴随状语） Without anything left in the with结构是许多英 语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 二、with结构的用法 with是介词，其意义颇多，一时难掌握。为帮助大家理清头绪，以教材中的句子为例，进行分类，并配以简单的解释。在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件（详见上述例句）。 1.带着，牵着…… (表动作特征)。如： Run with the kite like this.

with用法归纳

with用法归纳 （1）“用……”表示使用工具，手段等。例如： ①We can walk with our legs and feet. 我们用腿脚行走。 ②He writes with a pencil. 他用铅笔写。 （2）“和……在一起”，表示伴随。例如： ①Can you go to a movie with me? 你能和我一起去看电影'>电影吗？ ②He often goes to the library with Jenny. 他常和詹妮一起去图书馆。 （3）“与……”。例如： I’d like to have a talk with you. 我很想和你说句话。 （4）“关于，对于”，表示一种关系或适应范围。例如： What’s wrong with your watch? 你的手表怎么了？ （5）“带有，具有”。例如： ①He’s a tall kid with short hair. 他是个长着一头短发的高个子小孩。 ②They have no money with them. 他们没带钱。 （6）“在……方面”。例如： Kate helps me with my English. 凯特帮我学英语。 （7）“随着，与……同时”。例如： With these words, he left the room. 说完这些话，他离开了房间。 [解题过程] with结构也称为with复合结构。是由with+复合宾语组成。常在句中做状语，表示谓语动作发生的伴随情况、时间、原因、方式等。其构成有下列几种情形： 1.with+名词(或代词)+现在分词 此时，现在分词和前面的名词或代词是逻辑上的主谓关系。 例如:1)With prices going up so fast, we can't afford luxuries. 由于物价上涨很快，我们买不起高档商品。（原因状语） 2)With the crowds cheering, they drove to the palace. 在人群的欢呼声中，他们驱车来到皇宫。（伴随情况） 2.with+名词(或代词)+过去分词 此时，过去分词和前面的名词或代词是逻辑上的动宾关系。

独立主格with用法小全

独立主格篇 独立主格，首先它是一个“格”，而不是一个“句子”。在英语中任何一个句子都要有主谓结构，而在这个结构中，没有真正的主语和谓语动词，但又在逻辑上构成主谓或主表关系。独立主格结构主要用于描绘性文字中，其作用相当于一个状语从句，常用来表示时间、原因、条件、行为方式或伴随情况等。除名词/代词+名词、形容词、副词、非谓语动词及介词短语外，另有with或without短语可做独立主格，其中with可省略而without不可以。*注：独立主格结构一般放在句首，表示原因时还可放在句末；表伴随状况或补充说明时，相当于一个并列句，通常放于句末。 一、独立主格结构： 1. 名词/代词+形容词 He sat in the front row, his mouth half open. Close to the bank I saw deep pools, the water blue like the sky. 靠近岸时，我看见几汪深池塘，池水碧似蓝天。 2. 名词/代词+现在分词 Winter coming, it gets colder and colder. The rain having stopped, he went out for a walk.

The question having been settled, we wound up the meeting. 也可以The question settled, we wound up the meeting. 但含义稍有差异。前者强调了动作的先后。 We redoubled our efforts, each man working like two. 我们加倍努力，一个人干两个人的活。 3. 名词/代词+过去分词 The job finished, we went home. More time given, we should have done the job much better. *当表人体部位的词做逻辑主语时，不及物动词用现在分词，及物动词用过去分词。 He lay there, his teeth set, his hands clenched, his eyes looking straight up. 他躺在那儿，牙关紧闭，双拳紧握，两眼直视上方。 4. 名词/代词+不定式 We shall assemble at ten forty-five, the procession to start moving at precisely eleven. We divided the work, he to clean the windows and I to sweep the floor.

with复合宾语的用法(20201118215048)

with+复合宾语的用法 一、with的复合结构的构成 二、所谓"with的复合结构”即是"with+复合宾语”也即"with +宾语+宾语补足语” 的结构。其中的宾语一般由名词充当（有时也可由代词充当）；而宾语补足语则是根据 具体的需要由形容词，副词、介词短语，分词短语（包括现在分词和过去分词）及不定式短语充当。下面结合例句就这一结构加以具体的说明。 三、1、with +宾语+形容词作宾补 四、①He slept well with all the windows open.（82 年高考题） 上面句子中形容词open作with的宾词all the windows的补足语， ②It' s impolite to talk with your mouth full of food. 形容词短语full of food 作宾补。Don't sleep with the window ope n in win ter 2、with+宾语+副词作宾补 with Joh n away, we have got more room. He was lying in bed with all his clothes on. ③Her baby is used to sleeping with the light on.句中的on 是副词，作宾语the light 的补足语。 ④The boy can t play with his father in.句中的副词in 作宾补。 3、with+宾语+介词短语。 we sat on the grass with our backs to the wall. his wife came dow n the stairs,with her baby in her arms. They stood with their arms round each other. With tears of joy in her eyes ,she saw her daughter married. ⑤She saw a brook with red flowers and green grass on both sides. 句中介词短语on both sides 作宾语red flowersandgreen grass 的宾补， ⑥There were rows of white houses with trees in front of them.，介词短语in front of them 作宾补。 4、with+宾词+分词（短语 这一结构中作宾补用的分词有两种，一是现在分词，二是过去分词，一般来说，当分词所表 示的动作跟其前面的宾语之间存在主动关系则用现在分词，若是被动关系，则用过去分词。 ⑦In parts of Asia you must not sit with your feet pointing at another person.（高一第十课），句中用现在分词pointing at…作宾语your feet的补足语，是因它们之间存在主动关系，或者说point 这一动作是your feet发出的。 All the after noon he worked with the door locked. She sat with her head bent. She did not an swer, with her eyes still fixed on the wall. The day was bright,with a fresh breeze（微风）blowing. I won't be able to go on holiday with my mother being ill. With win ter coming on ,it is time to buy warm clothes. He soon fell asleep with the light still bur ning. ⑧From space the earth looks like ahuge water covered globe,with a few patches of land stuk ing out above the water而在下面句子中因with的宾语跟其宾补之间存在被动关系，故用过去分词作宾补：

with用法小结

with用法小结 一、with表拥有某物 Mary married a man with a lot of money . 马莉嫁给了一个有着很多钱的男人。 I often dream of a big house with a nice garden . 我经常梦想有一个带花园的大房子。 The old man lived with a little dog on the lonely island . 这个老人和一条小狗住在荒岛上。 二、with表用某种工具或手段 I cut the apple with a sharp knife . 我用一把锋利的刀削平果。 Tom drew the picture with a pencil . 汤母用铅笔画画。 三、with表人与人之间的协同关系 make friends with sb talk with sb quarrel with sb struggle with sb fight with sb play with sb work with sb cooperate with sb I have been friends with Tom for ten years since we worked with each other, and I have never quarreled with him . 自从我们一起工作以来，我和汤姆已经是十年的朋友了，我们从没有吵过架。 四、with 表原因或理由 John was in bed with high fever . 约翰因发烧卧床。 He jumped up with joy . 他因高兴跳起来。 Father is often excited with wine . 父亲常因白酒变的兴奋。 五、with 表“带来”，或“带有,具有”，在…身上，在…身边之意

(完整版)with的复合结构用法及练习

with复合结构 一. with复合结构的常见形式 1.“with+名词/代词+介词短语”。 The man was walking on the street, with a book under his arm. 那人在街上走着，腋下夹着一本书。 2. “with+名词/代词+形容词”。 With the weather so close and stuffy, ten to one it’ll rain presently. 天气这么闷热，十之八九要下雨。 3. “with+名词/代词+副词”。 The square looks more beautiful than even with all the light on. 所有的灯亮起来，广场看起来更美。 4. “with+名词/代词+名词”。 He left home, with his wife a hopeless soul. 他走了，妻子十分伤心。 5. “with+名词/代词+done”。此结构过去分词和宾语是被动关系，表示动作已经完成。 With this problem solved, neomycin 1 is now in regular production. 随着这个问题的解决，新霉素一号现在已经正式产生。 6. “with+名词/代词+-ing分词”。此结构强调名词是-ing分词的动作的发出者或某动作、状态正在进行。 He felt more uneasy with the whole class staring at him. 全班同学看着他，他感到更不自然了。 7. “with+宾语+to do”。此结构中，不定式和宾语是被动关系，表示尚未发生的动作。 So in the afternoon, with nothing to do, I went on a round of the bookshops. 由于下午无事可做，我就去书店转了转。 二. with复合结构的句法功能 1. with 复合结构，在句中表状态或说明背景情况，常做伴随、方式、原因、条件等状语。With machinery to do all the work, they will soon have got in the crops. 由于所有的工作都是由机器进行，他们将很快收完庄稼。（原因状语） The boy always sleeps with his head on the arm. 这个孩子总是头枕着胳膊睡觉。（伴随状语）The soldier had him stand with his back to his father. 士兵要他背对着他父亲站着。（方式状语）With spring coming on, trees turn green. 春天到了，树变绿了。（时间状语） 2. with 复合结构可以作定语 Anyone with its eyes in his head can see it’s exactly like a rope. 任何一个头上长着眼睛的人都能看出它完全像一条绳子。 【高考链接】 1. ___two exams to worry about, I have to work really hard this weekend.（04北京） A. With B. Besides C. As for D. Because of 【解析】A。“with+宾语+不定式”作状语，表示原因。 2. It was a pity that the great writer died, ______his works unfinished. (04福建) A. for B. with C. from D.of 【解析】B。“with+宾语+过去分词”在句中作状语，表示状态。 3._____production up by 60%, the company has had another excellent year. (NMET) A. As B.For C. With D.Through 【解析】C。“with+宾语+副词”在句中作状语，表示程度。

With复合结构的用法小结

With复合结构的用法小结 with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二 部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词； 2. with或without-名词/代词+副词； 3. with或without-名词/代词+介词短语； 4. with或without-名词/代词+动词不定式； 5. with或without-名词/代词+分词。 下面分别举例： 1、She came into the room，with her nose red because of cold.（with+名词+形容词，作伴随状语） 2、With the meal over ，we all went home.（with+名词+副词，作时间状语） 3、The master was walking up and down with the ruler under his arm。（with+名词+介词短语，作伴随状语。）The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.（with+名词+不定式，作伴随状语）He could not finish it without me to help him.（without+代词+不定式，作条件状语） 5、She fell asleep with the light burning.（with+名词+现在分词，作伴随状语）Without anything left in the cupboard，shewent out to get something to eat.（without+代词+过去分词，作为原因状语） 二、with结构的用法 在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件（详见上述例句）。 With结构在句中也可以作定语。例如： 1.I like eating the mooncakes with eggs. 2.From space the earth looks like a huge water-covered globe with a few patches of land sticking out above the water. 3.A little boy with two of his front teeth missing ran into the house. 三、with结构的特点 1. with结构由介词with或without+复合结构构成。复合结构中第一部分与第二部分语法上是宾语和宾语补足语关系，而在逻辑上，却具有主谓关系，也就是说，可以用第一部分作主语，第二部分作谓语，构成一个句子。例如：With him taken care of，we felt quite relieved.（欣慰）→（He was taken good care of.）She fell asleep with the light burning. →（The light was burning.）With her hair gone，there could be no use for them. →（Her hair was gone.） 2. 在with结构中，第一部分为人称代词时，则该用宾格代词。例如：He could not finish it without me to help him. 四、几点说明： 1. with结构在句子中的位置：with 结构在句中作状语，表示时间、条件、原因时一般放在

with的用法

with[wIT] prep.1.与…(在)一起，带着：Come with me. 跟我一起来吧。/ I went on holiday with my friend. 我跟我朋友一起去度假。/ Do you want to walk home with me? 你愿意和我一道走回家吗 2.(表带有或拥有)有…的，持有，随身带着：I have no money with me. 我没有带钱。/ He is a man with a hot temper. 他是一个脾气暴躁的人。/ We bought a house with a garden. 我们买了一座带花园的房子。/ China is a very large country with a long history. 中国是一个具有历史悠久的大国。3.(表方式、手段或工具)以，用：He caught the ball with his left hand. 他用左手接球。/ She wrote the letter with a pencil. 她用铅笔写那封信。4.(表材料或内容)以，用：Fill the glass with wine. 把杯子装满酒。/ The road is paved with stones. 这条路用石头铺砌。5.(表状态)在…的情况下，…地：He can read French with ease. 他能轻易地读法文。/ I finished my homework though with difficulty. 虽然有困难，我还是做完了功课。6.(表让步)尽管，虽然：With all his money, he is unhappy. 尽管他有钱，他并不快乐。/ With all his efforts, he lost the match. 虽然尽了全力，他还是输了那场比赛。7.(表条件)若是，如果：With your permission, I’ll go. 如蒙你同意我就去。8.(表原因或理由)因为，由于：He is tired with work. 他工作做累了。/ At the news we all jumped with joy. 听到这消息我们都高兴得跳了起来。9.(表时间)当…的时候，在…之后：With that remark, he left. 他说了那话就离开了。/ With daylight I hurried there to see what had happened. 天一亮我就去那儿看发生了什么事。10. (表同时或随同)与…一起，随着：The girl seemed to be growing prettier with each day. 那女孩好像长得一天比一天漂亮。11.(表伴随或附带情况)同时：I slept with the window open. 我开着窗户睡觉。/ Don’t speak with your mouth full. 不要满嘴巴食物说话。12.赞成，同意：I am with you there. 在那点上我同你意见一致。13.由…照看，交…管理，把…放在某处：I left a message for you with your secretary. 我给你留了个信儿交给你的秘书了。/ The keys are with reception. 钥匙放在接待处。14 (表连同或包含)连用，包含：The meal with wine came to ￡8 each. 那顿饭连酒每人8英镑。/ With preparation and marking a teacher works 12 hours a day. 一位老师连备课带批改作业每天工作12小时。15. (表对象或关系)对，关于，就…而言，对…来说：He is pleased with his new house. 他对他的新房子很满意。/ The teacher was very angry with him. 老师对他很生气。/ It’s the same with us students. 我们学生也是这样。16.(表对立或敌对)跟，以…为对手：The dog was fighting with the cat. 狗在同猫打架。/ He’s always arguing with his brother. 他老是跟他弟弟争论。17.(在祈使句中与副词连用)：Away with him! 带他走！/ Off with your clothes! 脱掉衣服！/ Down with your money! 交出钱来！ 【用法】1.表示方式、手段或工具等时(=以，用)，注意不要受汉语意思的影响而用错搭配，如“用英语”习惯上用in English，而不是with English。2.与某些抽象名词连用时，其作用相当于一个副词：with care=carefully 认真地/ with kindness=kindly 亲切地/ with joy=joyfully 高兴地/ with anger=angrily 生气地/ with sorrow=sorrowfully 悲伤地/ with ease=easily 容易地/ with delight=delightedly 高兴地/ with great fluency =very fluently 很流利地3.表示条件时，根据情况可与虚拟语气连用：With more money I would be able to buy it. 要是钱多一点，我就买得起了。/ With better equipment, we could have finished the job even sooner. 要是设备好些，我们完成这项工作还要快些。4.比较with 和as：两者均可表示“随着”，但前者是介词，后者是连词：He will improve as he grows older. 随着年龄的增长，他会进步的。/ People’s ideas change with the change of the times. 时代变了，人们的观念也会变化。5.介词with和to 均可表示“对”，但各自的搭配不同，注意不要受汉语意思的影响而用错，如在kind, polite, rude, good, married等形容词后通常不接介词with而接to。6.复合结构“with+宾语+宾语补足语”是一个很有用的结构，它在句中主要用作状语，表示伴随、原因、时间、条件、方式等；其中的宾语补足语可以是名词、形容词、副词、现在分词、过去分词、不定式、介词短语等：I went out with the windows open. 我外出时没有关窗户。/ He stood before his teacher with his head down. 他低着头站在老师面前。/ He was lying on the bed with all his clothes on. 他和衣躺在床上。/ He died with his daughter yet a schoolgirl. 他去世时，女儿还是个小学生。/ The old man sat there with a basket beside her. 老人坐在那儿，身边放着一个篮子。/ He fell asleep with the lamp burning. 他没熄灯就睡着了。/ He sat there with his eyes closed. 他闭目坐在那儿。/ I can’t go out with all these clothes to wash. 要洗这些衣服，我无法出去了。这类结构也常用于名词后作定语：The boy with nothing on is her son. 没穿衣服的这个男孩子是她儿子。 （摘自《英语常用词多用途词典》金盾出版社） - 1 -

with_的复合结构

with without 引导的独立主格结构 介词with without +宾语+宾语的补足语可以构成独立主格结构，上面讨论过的独立主格结构的几种情况在此结构中都能体现。 A．with+名词代词+形容词 He doesn’t like to sleep with the windows open. 他不喜欢开着窗子睡觉。 = He doesn’t like to sleep when the windows are open. He stood in the rain, with his clothes wet. 他站在雨中，衣服湿透了。 = He stood in the rain, and his clothes were wet. 注意： 在“with+名词代词+形容词”构成的独立主格结构中，也可用已形容词化的-ing 形式或-ed形式。 With his son so disappointing，the old man felt unhappy. 由于儿子如此令人失望,老人感到很不快乐。 With his father well-known, the boy didn’t want to study. 父亲如此出名，儿子不想读书。 B．with+名词代词+副词 Our school looks even more beautiful with all the lights on. 所有的灯都打开时，我们的学校看上去更美。 = Our school looks even more beautiful if when all the lights are on. The boy was walking, with his father ahead. 父亲在前，小孩在后走着。 = The boy was walking and his father was ahead. C．with+名词代词+介词短语 He stood at the door, with a computer in his hand. 或 He stood at the door, computer in hand. 他站在门口，手里拿着一部电脑。 = He stood at the door, and a computer was in his hand. Vincent sat at the desk, with a pen in his mouth. 或 Vincent sat at the desk, pen in mouth. 文森特坐在课桌前，嘴里衔着一支笔。 = Vincent sat at the desk, and he had a pen in his mouth. D．with+名词代词+动词的-ed形式 With his homework done, Peter went out to play. 作业做好了，彼得出去玩了。 = When his homework was done, Peter went out to play. With the signal given, the train started. 信号发出了，火车开始起动了。 = After the signal was given, the train started. I wouldn’t dare go home without the job finished. 工作还没完成，我不敢回家。 = I wouldn’t dare go home because the job was not finished.

with的复合结构用法及练习

页眉内容 with复合结构 一. with复合结构的常见形式 1.“with+名词/代词+介词短语”。 The man was walking on the street, with a book under his arm. 那人在街上走着，腋下夹着一本书。 2. “with+名词/代词+形容词”。 With the weather so close and stuffy, ten to one it’ll rain presently. 天气这么闷热，十之八九要下雨。 3. “with+名词/代词+副词”。 The square looks more beautiful than even with all the light on. 所有的灯亮起来，广场看起来更美。 4. “with+名词/代词+名词”。 He left home, with his wife a hopeless soul. 他走了，妻子十分伤心。 5. “with+名词/代词+done”。此结构过去分词和宾语是被动关系，表示动作已经完成。 With this problem solved, neomycin 1 is now in regular production. 随着这个问题的解决，新霉素一号现在已经正式产生。 6. “with+名词/代词+-ing分词”。此结构强调名词是-ing分词的动作的发出者或某动作、状态正在进行。 He felt more uneasy with the whole class staring at him. 全班同学看着他，他感到更不自然了。7. “with+宾语+to do”。此结构中，不定式和宾语是被动关系，表示尚未发生的动作。 So in the afternoon, with nothing to do, I went on a round of the bookshops. 由于下午无事可做，我就去书店转了转。 二. with复合结构的句法功能 1. with 复合结构，在句中表状态或说明背景情况，常做伴随、方式、原因、条件等状语。With machinery to do all the work, they will soon have got in the crops. 由于所有的工作都是由机器进行，他们将很快收完庄稼。（原因状语） The boy always sleeps with his head on the arm. 这个孩子总是头枕着胳膊睡觉。（伴随状语）The soldier had him stand with his back to his father. 士兵要他背对着他父亲站着。（方式状语）With spring coming on, trees turn green. 春天到了，树变绿了。（时间状语） 2. with 复合结构可以作定语 Anyone with its eyes in his head can see it’s exactly like a rope. 任何一个头上长着眼睛的人都能看出它完全像一条绳子。 【高考链接】 1. ___two exams to worry about, I have to work really hard this weekend.（04北京） A. With B. Besides C. As for D. Because of 【解析】A。“with+宾语+不定式”作状语，表示原因。 2. It was a pity that the great writer died, ______his works unfinished. (04福建) A. for B. with C. from D.of 【解析】B。“with+宾语+过去分词”在句中作状语，表示状态。 3._____production up by 60%, the company has had another excellent year. (NMET) A. As B.For C. With D.Through 【解析】C。“with+宾语+副词”在句中作状语，表示程度。

with用法

with结构是许多英语复合结构中最常用的一种。学好它对学好复合宾语结构、不定式复合结构、动名词复合结构和独立主格结构均能起很重要的作用。本文就此的构成、特点及用法等作一较全面阐述，以帮助同学们掌握这一重要的语法知识。 一、with结构的构成 它是由介词with或without+复合结构构成，复合结构作介词with或without的复合宾语，复合宾语中第一部分宾语由名词或代词充当，第二部分补足语由形容词、副词、介词短语、动词不定式或分词充当，分词可以是现在分词，也可以是过去分词。With结构构成方式如下： 1. with或without-名词/代词+形容词； 2. with或without-名词/代词+副词； 3. with或without-名词/代词+介词短语； 4. with或without-名词/代词+动词不定式； 5. with或without-名词/代词+分词。 下面分别举例： 1、She came into the room，with her nose red because of cold.（with+名词+形容词，作伴随状语） 2、With the meal over ，we all went home.（with+名词+副词，作时间状语） 3、The master was walking up and down with the ruler under his arm。（with+名词+介词短语，作伴随状语。）The teacher entered the classroom with a book in his hand. 4、He lay in the dark empty house，with not a man ，woman or child to say he was kind to me.（with+名词+不定式，作伴随状语）He could not finish it without me to help him.（without+代词+不定式，作条件状语） 5、She fell asleep with the light burning.（with+名词+现在分词，作伴随状语） 6、Without anything left in the cupboard，she went out to get something to eat.（without+代词+过去分词，作为原因状语） 二、with结构的用法 在句子中with结构多数充当状语，表示行为方式，伴随情况、时间、原因或条件（详见上述例句）。