High-Resolution Tandem Mass Spectrometry Qhia Txog Tus Qauv Tshwj Xeeb Ganglioside hauv Early Diabetic raum Kab Mob Ntawm Hom 2 Mob ntshav qab zib mellitus Cov neeg mob Ⅱ

2.2. Cov ncauj lus kom ntxaws txog kev txheeb xyuas ntawm polysialylated Species Associated to Macroalbuminuria los ntawm HCD MS / MS

HR-MS kev tshuaj ntsuam tau pom tias ganglioside ntawm cov neeg mob macroalbuminuric yog tus cwj pwm los ntawm kev nce siab tag nrho sialic acid cov ntsiab lus. Tsis pub tsawg tshaj plaub pentasialo gangliotetraoses ntawm GQ1 chav kawm tau pom nyob rau hauv cov qauv A3 uas muaj qhov tseeb loj. Lub ion ntawm m/z 812.7{{10}}}}68, kuaj pom tsuas yog nyob rau hauv kev soj ntsuam huab hwm coj spectrum ntawm A3, raug muab, raws li huab hwm coj xam, rau triply deprotonated thiab sodiated GQ1(d18:1/18 :0). Txhawm rau txhawm rau txheeb xyuas cov kab mob no cuam tshuam nrog macroalbuminuria kom ntxaws, peb cais cov ion ntawm m / z 812.7068 thiab xa mus rau HCD MS / MS ntawm HR hauv hom tsis zoo ion. Lub hom phiaj ntawm qhov kev sim MS tandem no yog: (i) kev tshawb xyuas ntawm cov qauv oligosaccharide saw; (ii) qhov kev pom zoo ntawm cov tshuaj ceramide muaj pes tsawg leeg, uas yog postulated xav txog qhov loj ntawm tag nrho cov GQ1 molecules; (iii) khaws cov ntaub ntawv tshwj xeeb raws li qhov chaw nyob ntawm Neu5Ac monosaccharides raws lub pob txha oligosaccharide, ua rau muaj kev ntxub ntxaug los ntawm tsib feem ntau GQ1 (d18: 1/18: 0) cov qauv (Daim duab 3) uas tej zaum yuav muaj nyob rau hauv cov zis ntawm cov neeg mob macroalbuminuric.

Lub fragmentation spectrum ntawm [M-4H+Na]3- precursor ion kuaj pom ntawm m/z 812.7068, generated los ntawm combining TIC tau rau ob feeb nyob rau hauv sib txawv zog sib tsoo zog nyob rau hauv ib tug 30-80 eV ntau yam, yog depicted hauv daim duab 4.

Daim duab 3. tsib feem ntau isomers ntawm GQ1(d18:1/18:0): (A) GQ1a(d18:1/18:{{10}}}) iso mer; (B) GQ1b(d18:1/18:{{20}}}) isomer; (C) GQ1c(d18:1/18:0)isomer; (D) GQ1d(d18:1/18:0) isomer; (E) GQ1e(d18:1/18:0) isomer.

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Nyem qhov no kom tau txais Natural organic CISTANCHE EXTRACT nrog 25% ECHINACOSIDE thiab 9% ACTEOSIDE rau raum kab mob

Cov ntaub ntawv MS/MS validate (d18:1/18:0) muaj pes tsawg leeg ntawm ceramide los ntawm Y0 ntawm m/z 564.8704 thiab Y1 ntawm m/z 726.5845 sib raug rau Glc-Cer ib ntus, nrog rau cov cleavage ion, S, ntawm m/z 325.1828 sib xws rau C18: 0 fatty acid (Daim duab 5). Ib yam li ntawd, tag nrho sialylation xwm txheej ntawm cov molecule kuj tau sau tseg. Li no, los ntawm B1 ion kuaj pom ntawm m/z 290.0868 thiab nws cov neeg sib tw sodiated ntawm m/z 312.0686 sib haum mus rau detachment ntawm ib tug Neu5Ac residue los ntawm niam txiv ion, ua ke nrog lub distal keeb kuaj ntawm m/z 581.18063 thiab m/z. , paub meej tias qhov tshwm sim ntawm Neu5Ac-Neu5Ac txuas.

Daim duab 5. Scheme of the fragmentation by HCD MS/MS experienced by the [M-4H++}}Na+ ] 3− precursor at m/z 812.7068 and the sequence ions diagnostic for the GQ1d structural isomer.

Xav txog qhov feem ntau txuas txoj hauj lwm ntawm Neu5Ac residues ntawm sab hauv lossis sab nraud Gal, muaj tsib tus neeg tuaj yeem tsim qauv rau GQ1(d18:1/18:0), raws li qhia hauv daim duab 3. Txawm li cas los xij, cov ion ntawm m/z 1032.3662 tsim los ntawm ob daim ntawv cog lus thiab sab hauv lub nplhaib sib tsoo thiab muab rau Z4 / Y0/ 2,4A2, ua ke nrog cov ion kuaj pom ntawm m/z 936.7894 raws li Y2 / 3,5A1, txhawb kev txuas ntawm tag nrho plaub Neu5Ac residues rau sab hauv Gal. Txawm hais tias qhov xwm txheej ntawm lwm cov qauv isomers tsis tuaj yeem raug cais tawm tag nrho, cov khoom siv ob ntu no qhia tau tias cov qauv tsim qauv zoo ib yam nrog (D) tus neeg sib tw, uas sib haum rau d isomer ntawm GQ1 (d18: 1/18:0) , yog tam sim no nyob rau hauv cov qauv A3. Lub tswv yim nyob rau hauv daim duab 5 qhia txog txoj kev fragmentation uas tau ntsib los ntawm cov precursor ion sib raug rau GQ1d (d18: 1/18: 0).

Tom ntej no macroalbuminuria-associated polysialic tus neeg sib tw rau cov ncauj lus kom ntxaws kev soj ntsuam yog qhov [M-3H+] 3− ion kuaj pom tsuas yog hauv kev tshuaj ntsuam huab hwm coj ntawm A3 qauv ntawm m/z 708.3379 thiab muab, raws li kev xam pes tsawg, rau GT1(d18:1/18:0). Cov ion no raug cais tawm thiab xa mus rau qhov kev soj ntsuam fragmentation los ntawm tandem MS siv HCD. Cov khoom ion spectrum generated los ntawm summing up scans tau rau 2 min ntawm kev sib tsoo lub zog sib txawv ntawm 30 thiab 80 eV yog nthuav tawm nyob rau hauv daim duab 6, nrog rau cov qauv tshuaj ntawm cov hom no thiab cov hauj lwm ntawm cov cim tseem ceeb. Lub fragmentation mechanism raws li kev ua hauj lwm MS/MS tej yam kev mob, ua ke nrog cov diagnostic fragment ions, yog depicted nyob rau hauv daim duab 7. Txhawm rau txhim kho qhov pom ntawm tag nrho cov khoom ions, Table 3 ua tiav cov npe ntawm cov cim qhia hauv cov spectrum uas tau nthuav tawm hauv daim duab 6.

Daim duab 6. Kev txheeb xyuas tus qauv los ntawm (−) nanoESI HR HCD MS/MS ntawm [M-3H+] 3− precursor ion kuaj pom hauv A3 qauv ntawm m/z 708.3379, uas, raws li rau kev suav loj, sib raug rau GT1(d18:1/18:0). Lub sij hawm tau txais: 2 min; Kev sib tsoo sib txawv ntawm lub zog tsis pub dhau 30-80 eV. Inset: tus qauv ntawm GT1b(d18:1/18:0) isomer txiav los ntawm MS/MS cov ntaub ntawv.

HCD fragmentation tau nce mus rau ntau ntu ions, uas tshwm sim los ntawm glycosidic bonds thiab cross-ring cleavages. Cov ions no muaj txiaj ntsig zoo rau kev ua haujlwm ntawm tag nrho cov carbohydrate ib ntus, nrog rau kev txheeb xyuas ntawm GT1 txoj haujlwm isomers, thiab rau kev pom zoo ntawm ceramide aglycone muaj pes tsawg leeg.

Lub d18:1/18:0 hom ceramide tau tshwm sim los ntawm Y0 ion ntawm m/z 564.5353 thiab txhawb nqa los ntawm Glc-Cer ib ntus txheeb xyuas los ntawm Y1 ntawm m/z 726.5879. Tsis tas li ntawd, txoj haujlwm ion ua pov thawj pom muaj GT1 isoform. Lub ion ntawm m/z 493.1669, nrog ib tug NeuAc-GalNAc muaj pes tsawg leeg, yuav tshwm sim tsuas yog los ntawm GT1, uas yog yus muaj los ntawm lub sialylation ntawm GalNAc residue. Cov seem ntawm cov ions fragment qhia qhov muaj nyob ntawm GT1b cov qauv isomer hauv A3 qauv. Cov qauv ntawm GT1b tau txais kev txhawb nqa los ntawm tag nrho cov ions tshwm sim los ntawm qhov tsis txo qhov kawg ntawm cov molecule, sau tseg los ntawm Y-hom series, nrog rau ob Z ions, uas ib qho tshwm sim hauv qis dua (Daim duab 6). Txawm li cas los xij, Y2 / B2 tau kuaj pom tias yog ib qho ion ntawm kev ncaj ncees ntau ntawm m / z 888.6405 muab pov thawj ntawm Gal-Glc-Cer ib ntus, tejzaum nws tshwm sim los ntawm GT1a lossis GT1b isomer tom qab qhov desialylation los ntawm Neu5Ac lossis Neu5Ac-Neument5Ac sab hauv Gal los yog los ntawm ib qho isomer uas tsis yog hloov sab hauv Gal, xws li GT1d.

Ntawm qhov tod tes, Y2 kuaj pom ntawm m/z 734.5701 substantiates lub disialo Neu5AcNeu5Ac keeb hauv zos nyob rau sab hauv Gal, ib qho kev teeb tsa zoo ib yam nrog GT1b isomer. Lub xub ntiag ntawm cov pab pawg distal kuj tseem muaj pov thawj los ntawm B2 ion ntawm m / z 581.1828.

Nyob rau hauv HCD MS / MS, ntau lub teeb liab muaj feem xyuam rau ib feem ntawm cov molecule desialylation, xws li Y4 thiab / los yog Y3 ions sib raug rau cov desialylated Gg4Cer ib ntus; Txawm li cas los xij, cov ions no tsis tuaj yeem qhia qhov chaw txuas ntawm Neu5Ac ntawm nruab nrab Gg4 glycan core. Tsis tas li ntawd, lub teeb liab ntawm m / z 364.1243 sib raug rau Gal-GalNAc ib ntus muab rau GT1b sab hauv cleavage B3 / B1 ion yog vim li cas rau GT1c isomer vim tias qhov glycoform no muaj qhov tsis hloov pauv lub davhlau ya nyob twg Gal-GalNAc disaccharide. Li no, tshwj tsis yog rau Y2 , uas cuam tshuam rau GT1b, tag nrho cov ions tuaj yeem tshwm sim los ntawm lwm yam Neu5Ac txoj haujlwm isomers, xws li GT1a, GT1, GT1c, lossis txawm tias tsis tshua muaj qhia GT1d. Ntawm qhov tod tes, Y2 nws tus kheej tuaj yeem yog qhov tshwm sim ntawm qhov tshwm sim tsis tshua muaj tshwm sim, uas tseem tshem tawm Neu5Ac los ntawm cov kev sim sib txuas nrog rau sab hauv Gal.

Muab qhov ntawd, tshwj tsis yog kev tshaj tawm GT1, MS / MS cov ntaub ntawv ntsig txog glycosidic daim ntawv cog lus tsis tuaj yeem ua rau tsis meej pem rau lwm cov GT1 isomers hauv A3 sib tov, peb ua tib zoo tshuaj xyuas lub nplhaib cleavage ions txhawm rau txhawm rau tshawb pom muaj peev xwm sab hauv fragmentation txhawb GT1b. Peb pom qhov tsawg-siv triply them ion ntawm m/z 634.6067, uas zoo ib yam nrog 0,2 hom cross-ring cleavage ntawm ib qho ntawm lub davhlau ya nyob twg sialic acid residues, uas, hauv cov ntaub ntawv ntawm GT1b hom, tuaj yeem tshwm sim los ntawm 0, 2X4 lossis 0,2X3 lossis ob qho tib si. Vim tias GT1b isomer feem ntau muaj ntau dua li GT1, Daim duab 6 thiab 7 nthuav tawm cov haujlwm ion thiab cov txheej txheem fragmentation tshwj xeeb rau GT1b.

Vim yog Gal-GalNAc-Gal saw symmetry, tsis muaj peev xwm txiav txim siab tag nrho lwm cov qauv isomers. Txawm hais tias HCD MS/MS cov ntaub ntawv qhia txog qhov muaj nyob ntawm GQ1d(d18:1/18:0), GT1 (d18:1/18:0), thiab GT1b(d18:1/18: 0) hauv A3 qauv, qhov tshwm sim ntawm lwm yam GQ1 thiab GT1 isomers tsis tuaj yeem raug cais tawm. Txawm li cas los xij, peb qhov kev tshawb pom qhia tias peb isomers-GQ1d(d18:1/18:0), GT1b(d18:1/18:{{30}}), thiab GT1 (d18:1) / 18: 0) yog tam sim no nyob rau hauv cov qauv A3 thiab tej zaum yuav tau kawm ntxiv raws li cov cim molecular ntawm macroalbuminuria.

3. Cov ntaub ntawv thiab cov txheej txheem

3.1. Yam Kev Sau Npe Kawm Ntawv

Txhawm rau txhim kho thiab siv tau txoj hauv kev, ib qho kev tshuaj ntsuam xyuas ntawm hom 2 DM cov neeg mob thiab cov kev tswj hwm kev noj qab haus huv tau raug soj ntsuam hauv kev tshawb fawb hla ntu ntu. Tag nrho ntawm 30 hom 2 DM cov neeg mob tuaj koom Lub Tsev Haujlwm Saib Xyuas Kev Mob Sab Nraud ntawm Nephrology thiab Lub Tsev Haujlwm Saib Xyuas Kev Mob Sab Nraud ntawm Mob Ntshav Qab Zib thiab Kab Mob Metabolic tau muab faib ua 3 pawg raws litso zis albumin/creatinine piv(UACR) raws li hauv qab no: 10 cov neeg mob uas muaj normoalbuminuria (A1, txhais tias UACR <30 mg/g), 10 nrog microalbuminuria (A2, UACR 30–300 mg/g), thiab 10 nrog macroalbuminuria (A3, UACR> 300 mg/ g). Kaum hnub nyoog- thiab poj niam txiv neej-matched kev noj qab haus huv cov ntsiab lus (C) mus koom ib tug kws kho mob lub chaw ua hauj lwm rau niaj hnub tshuaj xyuas yam tsis paub txog keeb kwm ntawm lub raum kab mob thiab tsis muaj DM (tsis suav los ntawm tus nqi ntawm HbA1c tsawg dua lossis sib npaug li 5.6%) kuj tau tso npe rau hauv qhov no. kawm

Txhua tus neeg mob uas muaj nyob rau hauv txoj kev tshawb no muaj qhov tsawg kawg nkaus 5- xyoo ntawm DM, ruaj khov raum ua haujlwm tsawg kawg 2 xyoos, tswj ntshav siab (<130/80 mmHg), negative urinary sediment, and a negative urine culture. The exclusion criteria were represented by other causes of proteinuria (other glomerular diseases, neoplasia, or autoimmune diseases), hematuria, poor control of DM (HbA1c > 10%), liver diseases, and pregnant or lactating women. The patients had no indication for raum biopsy(tsis muaj hematuria thiab lwm yam ua rau muaj proteinuria, tsis muaj kev poob sai hauv GFR).

3.2. Nqe lus hais txog Ethics

Txoj kev tshawb no tau ua raws li Kev Tshaj Tawm ntawm Helsinki, thiab cov txheej txheem tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees hauv Kev Tshawb Fawb ntawm Lub Tsev Haujlwm (Board of Human Studies-"Victor Babes" University of Medicine and Pharmacy Timisoara, Nr. 15/12.09.2016 ; County Emergency Hospital, Nr. 100/23.11.2016). Txhua tus neeg mob thiab kev tswj hwm tau pom zoo koom nrog hauv txoj kev tshawb fawb los ntawm kev kos npe rau daim ntawv tso cai pom zoo.

3.3. Kev soj ntsuam kuaj

Cov zis ganglioside ntawm 30 Hom 2 DM cov neeg mob tau tshawb xyuas nyob rau hauv ib qho kev sib tw ua haujlwm los ntawm kev sib piv nrog 10 kev tswj xyuas kev noj qab haus huv. Biochemical parameters xa mus rau cov ntshav urea thiab creatinine, 24 teev proteinuria, UACR, tso zis sediment, thiab zis kab lis kev cai. Txhua tus neeg mob tsis zoo rau cov kab mob tso zis. Cov tshuaj muaj pes tsawg leeg ntawm cov tshuaj ganglioside yog kuaj pom los ntawm 24 teev cov zis kuaj. Cov zis cov zis ntawm cov neeg mob thiab kev tswj tau luv luv khaws cia ntawm −20 ◦C thiab thawed ua ntej kuaj.CKDtau txhais raws li KDIGO cov lus qhia rau kev soj ntsuam thiabtswj kev mob raum mob. Lub eGFR tau suav nrogmob raum mobKab mob sib kis sib koom ua ke sib npaug (CKD-EPI creatinine 2009 equation) [16].

3.4. Ganglioside Extraction thiab Purification

Ganglioside extraction ua raws li txoj kev tsim los ntawm Svennerholm thiab Fredman [17] thiab hloov kho los ntawm Vukeli'c li al. [18]. Peb yav dhau los tau hloov kho cov txheej txheem no rau kev tshem tawm thiab ntxuav cov gangliosides los ntawm cov kua hauv lub cev thiab siv rau hauv cov kua dej cerebrospinal (CSF) gangliosides. Cov txheej txheem tau piav qhia meej hauv peb txoj kev tshawb fawb yav dhau los ntsig txog profileing thiab fragmentation tsom xam ntawm CSF gangliosides los ntawm huab hwm coj spectrometry [19]. Luv luv, lipids tau muab rho tawm ob zaug siv chloroform (C) / methanol (M) / dej (W) sib tov rau tag nrho qhov sib piv ntawm 1: 2: 0.75 C / M / W, nrog "W" sib xws. tso zis; Yog li, qhov sib tov muaj 4 mL ntawm C, 8 mL ntawm M, thiab 3 mL ntawm cov zis. Analytical-qib chloroform thiab methanol tau yuav los ntawm Merck (Darmstadt, Lub Tebchaws Yelemees) thiab siv yam tsis muaj kev lim dej ntxiv. Tom qab ua tiav kev sib cais ntawm theem, ua raws li cov txheej txheem tau piav qhia hauv [19], cov theem sab saud uas muaj cov polar glycosphingolipids tau sau.

Kev lim dej ntawm cov khib nyiab khib nyiab tau ua tiav hauv ob peb kauj ruam [19]: tshem tawm cov precipitated protein- ntsev complexes, ua raws li centrifugation, gel pom ntawm Sephadex G-25 kem (Sigma-Aldrich, Burlington, MA, USA ) txhawm rau tshem tawm cov kab mob uas tsis tshua muaj molecular-yuag, thiab thaum kawg, kev lim ntshav hmo ntuj ntawm 4 ◦C tiv thaiv dej. Cov gangliosides tau muab rho tawm raws li cov xwm txheej zoo ib yam los ntawm tag nrho cov zis aliquots, tawm los ntawm A1 (normoalbuminuria), A2 (microalbuminuria), A3 (macroalbuminuria), thiab C (tswj) mixs.

3.5. Qauv Npaj rau Mass Spectrometry

Cov purified A1, A2, A3, thiab C ganglioside extracts tau evaporated kom tiav dryness hauv SpeedVac Concentrator SPD 111 V system (Savant, Düsseldorf, Lub teb chaws Yelemees) ua ke rau lub tshuab nqus tsev twj. Rau MS kev tshuaj ntsuam, txhua cov extract qhuav tau yaj hauv cov ntshiab methanol los tsim cov tshuaj hauv Tshuag thiab khaws cia ntawm −20 ◦C. Ua ntej MS tsom xam, cov kev daws teeb meem tau centrifuged hauv Sigma 2-16 qauv centrifuge (Sartorius AG, Göttingen, Lub teb chaws Yelemees). Rau Orbitrap MS infusion, ua hauj lwm cov qauv nrog ib tug concentration ntawm 5 pmol·µL −1 nyob rau hauv ntshiab methanol tau los ntawm diluting aliquots los ntawm cov Tshuag tov. Ganglioside concentration hauv cov tshuaj infused yog xam rau qhov nruab nrab molecular hnyav ntawm 2000 g mol-1.

3.6. Orbitrap Mass Spectrometry nrog nanoESI

Cov kev sim MS tau ua tiav ntawm LTQ Orbitrap Velos Pro ™ huab hwm coj spectrometer (Thermo Fisher Scientific, Bremen, Lub teb chaws Yelemees) nruab nrog offline nanoES qhov chaw ES 259 ​​(Thermo Fisher, Bremen, Lub teb chaws Yelemees).

Cov cuab yeej yog hybrid huab hwm coj spectrometer combining ob hom sib txawv ntawm huab cua analyzers thiab muab ntau yam zoo: siab rhiab heev thiab raug, zoo zoo ntawm cov ntaub ntawv thiab ceev ntawm kev tsom xam, lub peev xwm rau nrhiav tau ntawm me Cheebtsam nyob rau hauv complex mixs, tsawg tus qauv noj, thiab zam kev hla kev tshuaj xyuas thiab nqa los ntawm cov qauv mus rau cov qauv.

Tus thawj analyzer yog dual-siab linear quadrupole ion ntxiab muaj peev xwm cais cov ion nrog rau qhov tshwj xeeb-rau-them piv (m / z) thiab ua kom nws los ntawm kev muab lub zog kinetic rau hauv ion nrog rau qhov tshwj xeeb m / z hauv kev txiav txim. ua rau kev sib tsoo-induced dissociation. Qhov thib ob analyzer yog lub Orbitrap, uas muaj ib tug cylindrical txheej electrode thiab ib chim puab electrode, cuab cov ions nyob rau hauv lub voj voog ncig ntawm lub puab electrode. Nws muaj kev daws teeb meem zoo heev thiab muaj peev xwm ntawm kev sib txuas ntawm cov kab mob ionic hauv ntau theem MS (MSn) kev sim los ntawm cov txheej txheem fragmentation zoo.

Borosilicate capillaries (10} cm ntev) tau rub nrog Sutter p-97 micropipette ruber los tsim electrospray capillaries nrog 10 µm qhov ntau thiab tsawg thiab taper ntev ntawm 4 mm. Ib lub ntim ntawm 10 µL ntawm cov tshuaj ntawm ib tug concentration ntawm 5 pmol·µL −1 nyob rau hauv methanol tau nkag mus rau hauv lub nraub qaum ntawm lub emitter, thiab ib tug 0.25 hli platinum hlau tau muab tso rau hauv cov tshuaj. Cov txiaj ntsig muaj peev xwm siv rau cov hlau platinum thiab lub khob hliav qab tau hloov kho mus tas li kom ua tiav cov ionization zoo ntawm cov khoom. Cov txheej txheem nanoESI tau pib los ntawm kev teeb tsa cov ntsuas ntsuas raws li hauv qab no: nanoESI voltage, 0.80 kV; Lub cev muaj zog, 40-60V; desolvation kub, 80 ◦C; S-lens RF qib, 60%. Cov txiaj ntsig ntawm MS tsis tuaj yeem txhim kho cov ionization thiab cov tshuaj tsuag tsis tu ncua thiab, tib lub sijhawm, txo qis hauv qhov chaw tawg ntawm labile Neu5Ac residue txuas rau oligosaccharide core ntawm ganglioside molecule.

Tag nrho cov huab hwm coj loj (MS thiab tandem MS) tau txais hauv HR hom, kev kuaj pom tsis zoo ion hom, thiab nyob rau hauv thaj tsam 200–2000 m/z. MS scans tau txais nrog kev daws teeb meem rau 60,000. Qhov loj spectrometer tau ua haujlwm thiab tswj los ntawm LTQ Tune Plus v2.7 (Thermo Scientific, Bremen, Lub teb chaws Yelemees), thiab MS cov ntaub ntawv tau txais thiab ua tiav tau siv Xcalibur 3.0.63 software (Thermo Scientific, Bremen, Lub teb chaws Yelemees).

MS/MS kev sim tau ua hauv LTQ los ntawm CID hauv kev sib tsoo ntawm tes siv helium 5.0 purity ntawm lub siab ntawm 50 psi raws li cov roj sib tsoo. MS/MS scans tau txais nrog kev daws teeb meem rau 20,000. Kev xaiv ion thiab fragmentation tau ua manually. Lub precursor ions raug xaiv nyob rau hauv ib qho kev sib cais dav ntawm 1 m / z unit thiab fragmented los ntawm HCD siv sib txawv-kev sib tsoo energies nyob rau hauv lub 30-80 eV ntau yam los txhim kho cov kev pab cuam ntawm fragment ions.

Lub TICs thiab huab hwm coj spectra tau los ntawm kev sib txuas cov ntawv sau tau ua tiav siv Xcalibur 2.1 software (Thermo Scientific, Waltham, MA, USA), uas tso cai rau kev rho tawm ntawm cov spectra, nrog rau lawv cov smoothing thiab rho tawm.

Ua ntej qhov kev sim, qhov ntsuas m/z tau ntsuas sab nraud siv qhov kev siv siab ua lag luam hu ua Pierce® ESI Negative Ion Solution los ntawm Thermo Scientific (Waltham, MA, USA). Hauv hom tsis zoo ion, tus qauv no tau muab cov spectrum nrog kev ncaj ncees ionic kev pab cuam ntawm m / z range scanned hauv MS thiab tandem MS thwmsim.

Rau kev ua kom zoo ntawm nanoESI MS thiab MS / MS tej yam kev mob nyob rau hauv qhov tsis zoo ion hom, kev sib piv, thiab cov ntaub ntawv ntsuam xyuas, cov nram qab no tus qauv ganglioside fractions, uas muaj nyob rau hauv kev lag luam, tau ntsuas: GM1 (bovine hlwb); GD1a, GD1b, GT1b, thiab GQ1b (porcine hlwb); thiab GM3 thiab GD3 (bovine mis) los ntawm Avanti Polar Lipids (Birmingham, AL, USA), nrog rau tag nrho ganglioside extract los ntawm bovine hlwb, lag luam hu ua Cronassial sib tov, los ntawm Abano Terme (Padua, Ltalis).

3.7. Abbreviation ntawm Gangliosides

Rau kev ua haujlwm ganglioside, cov ntawv sau luv tau qhia hauv 1980 los ntawm Svenner-holm [20], nrog rau cov lus pom zoo los ntawm 1998 ntawm IUPAC-IUB Commission ntawm Biochemical Nomenclature, [21] tau siv raws li hauv qab no:

LacCer-Gal 4Glc 1Cer; GM3-II3 - -Neu5Ac-LacCer; GD3-II3 - -(Neu5Ac)2-LacCer; GT3-II3 - -(Neu5Ac)3-LacCer; GM2-II3 - -Neu5Ac-Gg3Cer; GD2-II3 - -(Neu5Ac)2-Gg3Cer; GM1a or GM1-II3 - -Neu5Ac-Gg4Cer; GM1b-IV3 - -Neu5Ac-Gg4Cer; GalNAc-GM1b-IV3 - -Neu5Ac-Gg5Cer; GD1a-IV3 - -Neu5Ac, II3 - -Neu5Ac-Gg4Cer; GD1b-II3 - -(Neu5Ac)2- Gg4Cer; GT1b-IV3 - -Neu5Ac, II3 - -(Neu5Ac)2-Gg4Cer; GQ1b-IV3 - -(Neu5Ac)2, II3 - - (Neu5Ac)2-Gg4Cer; nLM1 los yog 30 -nLM1-IV3 - -Neu5Ac-nLc4Cer; LM1 los yog 30 -isoLM1-IV3 - - Neu5Ac-Lc4Cer; nLD1-disialo-nLc4Cer.

3.8. MS Cov ntaub ntawv txhais lus

Vim tias tsis muaj cov software tshwj xeeb hauv computer, databases, lossis lwm yam kev pabcuam IT los pab txhais cov kev ntsuam xyuas ganglioside thiab tandem loj spectra muaj nyob rau niaj hnub no, hauv txoj kev tshawb no, cov tshuaj ions molecular tau raug xa mus rau ganglioside hom los ntawm kev xam pes tsawg pawg thiab raws li lub hauv paus. ntawm cov ntaub ntawv peb tau txais yav tas los ntawm hom glycosphingolipid thiab paub txog txoj hauv kev biosynthesis. Hauv kev txhais lus ntawm MS thiab MS / MS cov ntaub ntawv thiab kev suav loj, peb tau txais kev pabcuam los ntawm cov khoom lag luam loj ntawm ganglioside molecular thiab fragment ions uas peb tau txheeb xyuas thiab pom muaj ntau yam tib neeg cov ntaub so ntswg thiab cov kua dej rau hnub uas siv cov txheej txheem MS siab heev. Cov qauv no sawv cev rau peb cov ntaub ntawv qub, uas tau suav nrog hauv peb cov kev tshawb fawb yav dhau los [12,19,22–31]. Cov hom tshiab tau tshaj tawm hauv txoj haujlwm no ua tiav peb cov ntaub ntawv ganglioside tam sim no.

Txoj haujlwm ntawm oligosaccharide txha caj qaum ib ntus ions tsim nyob rau hauv HCD MS / MS ua raws li feem ntau lees txais nomenclature [24,25].

4. Cov lus xaus

Hauv txoj kev tshawb no, peb tau tsim ib txoj hauv kev raws li HR MS thiab tandem MS rau kev txiav txim siab ntawm ganglioside hauv DKD los ntawm kev sib piv ntawm cov zis tso zis los ntawm cov neeg mob normo, micro-, thiab macroalbuminuric, nrog rau kev tswj kev noj qab haus huv, ua raws li kev daws teeb meem zoo ib yam. thiab instrumental tej yam kev mob. Lub hom phiaj kawg ntawm peb txoj kev tshawb fawb yog los txiav txim seb qhov kev hloov pauv tshwm sim hauv qhov muaj pes tsawg leeg thiab cov qauv ntawm lub raum gangliosides qhia hauv DKD vs. tswj thiab nyob rau hauv ntau theem ntawm tus kab mob.

Lub siab rhiab heev, rov tsim dua tshiab, kev daws teeb meem, thiab qhov tseeb ntau yog muab los ntawm MS platform optimized rau lub hom phiaj no tau tso cai rau peb los tsim kom muaj sialylation degree ntawm cov hom uas tau hais los ua lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm tus kab mob, sawv cev rau tus cim ntawm DKD. , txawm nyob rau theem normoalbuminuria ntawm hom 2 DM cov neeg mob. Tsis tas li ntawd, cov kev hloov kho tau pom nyob rau hauv qhov muaj pes tsawg leeg ntawm Cer qhia tau hais tias, nyob ib sab ntawm cov qauv glycan, cov lipid moiety kuj tseem tuaj yeem suav tias yog cov ntiv tes molecular rau DKD theem.

Nyob rau theem siab dua ntawm kev tshawb fawb, MS / MS los ntawm HCD tau ua haujlwm rau kev txheeb xyuas cov qauv ntawm cov tsiaj nrog lub luag haujlwm biomarker. Nyob rau hauv cov ntaub ntawv ntawm tus qauv A3, cov molecular ions, uas, raws li cov huab hwm coj xam, tau pom muaj sib xws rau GQ1(d18:1/18:0) thiab GT1(d18:1/18:{{1 0}}), ntsig txog, raug cais tawm thiab xa mus rau kev sib cais hauv kev sib tsoo ntawm tes ntawm qhov ntsuas. Lub hom phiaj ntawm cov kev sim no yog los sau cov ntaub ntawv ntawm cov ncauj lus kom ntxaws kev teeb tsa ntawm cov molecule. Lub optimized sequencing tej yam kev mob, tshwj xeeb tshaj yog kev sib tsoo lub zog ntau yam thiab collisional gas siab, induced tshwj xeeb cleavages ntawm glycosidic bonds, uas ua rau cov tsim ntawm fragment ions pom raws li cov teeb meem nyob rau hauv lub tandem loj spectra. Ob peb ions tau pom muaj kev kuaj mob rau qee cov isomers ntsig txog qhov chaw ntawm Neu5Ac moieties hauv cov carbohydrate saw ntawm ganglioside molecule. Raws li cov ntaub ntawv qhia txog cov kab ke no, peb pom tias GQ1d(d18:1/18:0), GT1 (d18:1/18:0), thiab GT1b(d18:1/18: 0) isomers muaj nyob rau hauv cov qauv A3 thiab muaj feem xyuam rau macroalbuminuria.

Cov txiaj ntsig tau txais yuav tsum muaj kev lees paub ntxiv ntawm qee qhov kev xav hauv cov kev tshawb fawb ntev ua rau cov pab pawg loj dua txhawm rau ua pov thawj kev sib raug zoo ntawm kev sib raug zoo ntawm qhov nyuaj ntawm gangliosides kuaj pom hauv cov zis.Cov neeg mob DKDthiab kev cuam tshuam rau lub raum thaum ntxov hauv hom 2 DM, txawm tias nyob rau theem normoalbuminuria. Txawm li cas los xij, peb txiav txim siab tias qhov kev tshawb pom tam sim no qhib kev tshawb fawb txog kev tshawb nrhiav lub luag haujlwm hauv kev kis kab mob ua si los ntawm sialylation, O-acetylation, thiab O-fucosylation ntawm gangliosides, nrog rau lawv cov kev hloov kho los ntawm O-GalNAc thiab CH3COO-. Lwm qhov txiaj ntsig ntawm cov txiaj ntsig no yog kev txhim kho ntawm cov txheej txheem rau kev kuaj mob DKD thaum ntxov raws li ganglioside profiling siv cov khoom loj spectrometry.

Cov ntaub ntawv

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3. Lin, YC; Chang, YH; Yang, SY; Wu, KD; Chu, TS Update of pathophysiology and management of diabetes mellitus.J. Med. Assoc.2018, 117, 662–675. [CrossRef] [PubMed] 

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7. Kwak, DH; Rho, YI; Kwon, OD; Ahan, SH; Nkauj, JH; Chaw, YK; Kim, SJ; Choi, BK; Jung, KY Kev txo qis ntawm ganglioside GM3 hauv streptozocin-induced ntshav qab zib glomeruli ntawm nas.Lub neej Sci.2003, 72, 1997–2006. [CrossRef] 

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