(Università di Bologna)

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1 (Università di Bologna)
Accoppiamento della tecnica di frazionamento in campo flusso-flusso a fibra micro-tubulare porosa con spettrometria di massa electrospray per la caratterizzazione di proteine F. Dal Piaz, A. Zattoni, D. Parisi, M. Guardigli, P. Reschiglian, A. Roda (Università di Bologna)

2 Relazione Struttura/Funzione nelle molecole proteiche

3 ES/MS e conformazione proteica
Condizioni Denaturanti Condizioni Pseudo-Native A.E. Ashcroft et al (2002) J Biol Chem. 277(36):

4 ES/MS e complessi proteici
M. Fändrich, et al, (2000) Proc Natl Acad Sci U S A. 97(26):

5 Analytical Chemistry (2003)
Sistemi separativi classici e analisi on-line di proteine e complessi proteici in condizioni native RP-HPLC: Altamente denaturante Gel Filtration: Difficoltà di interfacciamento Elettroforesi Capillare: Blandamente denaturante Difficoltà di interfacciamento Analytical Chemistry (2003) In press

6 Principio della "Field-Flow Fractionation"
Introduzione del campione Campo esterno Lunghezza Flusso Spessore Larghezza Field-flow fractionation (FFF) is a family of separation techniques which are able to fractionate a broad range of macromolecules, nano- and micro-sized particles. It has been commonly referred to as “one-phase” chromatography since it uses a liquid mobile phase as in liquid chromatography. Nonetheless, the separation is structured inside a channel without stationary phase by the action of a field or gradient that is applied perpendicularly to the direction of the mobile phase flow. Classical FFF devices have been based on a flat, rectangular-shaped channel with capillary cross-section. Across the channel section the mobile phase profile is parabolic, as in capillary gas chromatographic columns. Typical channel dimensions are cm in length, 2 cm in breadth and micrometers in thickness. We can have different type of fields that define the different FFF variants which can be, consequently, applied to different type of samples. Detector Spessore Campione Flusso parabolico

7 Vrad Vin Vout Il canale HF FlFFF 1/8” PE fitting 1/8” PE Tee 1/8”
Teflon tube The idea of hollow fiber membranes as microcolumn channels for FlFFF was reported since However, high performance HF FlFFF of particles has been shown only recently by our Korean coworkers, through the optimization of the HF FlFFF channel and system design. In HF FlFFF sample separation is structured by the force generated by the radial flow through the porous wall of the HF membrane. In HF FlFFF the flow that is inlet into the HF channel is divided into two parts: part of the flow penetrates the HF inner wall as radial flow and the rest exits along the HF as axial flow. The microcolumn channels we have developed are made of polysulfone or chlorinated polyvinylchloride HF membranes. The HF dimensions are 24.0 cm in length and cm in nominal, inner radius. The HF is inserted into two pieces of 1/8” Teflon tube with a tee connection for the radial flow exit. Two 1/8” PEEK ferrules are used for the channel inlet and outlet connections to the system. The key advantage of this type of HF FlFFF channel clearly lies in its simplicity, low-cost and miniaturization, if compared to classical FlFFF systems. ……………. (fwd) …….. (bkw) These features allow for disposable usage of HF FlFFF. Disposable separators can be particularly appealing for analytical and micro-preparative scale bio-separations, in which either sterility or run-to-run reproducibility are critical. cPVC / PSf HF membrane 24x0.08 ID cm 1/8” PEEK ferrule 1/8” PEEK ferrule

8 Modo normale in HF FlFFF Meccanismo di ritenzione Browniano
Vrad z L Vout rf Flusso > < v Vin x U Campo

9 Frazionamento di proteine in HF FlFFF
Fase mobile: Ammonio acetato 50 mM ;pH7 Vin=0.70 ml/min ;Vrad=0.38 ml/min BSA 2% in FM ;mw 66 kDa Hrp 1% in FM ; mw40 kDa AP 1% in FM ; mw 160 kDa Fer 1% in FM ; mw 449 kDa

10 Costruzione della retta di taratura lnD lnM
vs lnM t R = — 8D ln V out in r 2 kT f D= D=A M - b 3 ph d -11,0 Fer -10,5 AP Y = A + b * X BSA -10,0 Hrp Hb ln D Parameter Value Error Mio -9,5 A - b - -9,0 -8,5 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0 13,5 ln M

11 HF FlFFF-ES/Q-TOF MS Valvola a 4 vie Valvola a spillo Manometro
0.75 0.50 Pompa 1 Valvola a spillo 1 2 3 Manometro Valvola a T Scarico 0.75 0.50 Pompa 2 Valvola d’iniezione Fase mobile Manometro UV-DAD 1 2 3 Fibra Valvola a spillo Fase mobile Scarico ES/Q-TOF Valvola di splitting

12 UV/vis DAD Valvola di splitting Canale HF FlFFF Sorgente ES

13 Analisi HF-FlFFF-MS della BSA
+55 +50 +45 +40 +36 +60 Peso Molecolare misurato: ±1.74 Da Peso Molecolare teorico: Da Fase mobile: Ammonio Acetato50mM ;pH7

14 Analisi HF-FlFFF-MS della HRP
B C D E F 80 70 60 50 nm(mAU) 40 30 20 10 2 4 6 8 10 12 1800 1600 1400 1200 1000 TIC 800 600 400 200 2 4 6 tempo (min) Fase mobile: Ammonio Acetato50mM ;pH7

15 Glicosilazione 2 Ca 2+ C 42343.1 D 42422.0 E 43165.0 F 43244.1 A
B 2 Ca 2+

16 Analisi HF-FlFFF-MS della mioglobina da cavallo
Fase mobile:ammonio acetato 50mM ;pH7 Analisi HF-FlFFF-MS della mioglobina da cavallo +9 +8 +7 +6 P. M. Mioglobina: Da P. M. Eme: Da P. M. Misurato: ±0.58 Da

17 HF-FlFFF-MS LC-MS A:17566.69±0.58 Da B:16951.48±0.27 Da A9 A8 A7 A10

18 Confronto spettri ES/MS del complesso Mioglobina/Eme
+9 +Na+ +2Na+ +3Na+ +CH3COO- Introduzione Diretta HF-FlFFF Confronto spettri ES/MS del complesso Mioglobina/Eme

19 Analisi HF-FlFFF-MS dell’emoglobina umana
P.M. teorico Hb-: Da P.M. teorico Hb-: Da Eme A Fase mobile: Ammonio Acetato50mM ;pH7

20 Conclusioni Prospettive
L’accoppiamento HF-FIFFF/spettrometria di massa costituisce un sistema semplice e robusto per l’analisi di proteine e complessi proteici in condizioni native: 1) L’interfacciamento non presenta problemi rilevanti. 2) L’assenza di interazione con una fase stazionaria e la possibilità di utilizzare qualsiasi tipo di solvente garantiscono il mantenimento della stabilita’ delle proteine e dei complessi in esame. Prospettive a) Miniaturizzazione del sistema separativo per interfacciamento diretto con sorgente nanospray b) Valutazione dell’efficienza nell’analisi di miscele più o meno complesse c) Possibilità di utilizzo dell’HF-FlFFF all’interno di un sistema cromatografico multidimensionale accoppiato con uno spettrometro di massa, per sudi nel campo della proteomica.

21 Ringraziamenti

22 å Efficenza in HF FlFFF + = H Focalizzazione Eluizione i poly long neq
inj Focalizzazione L0 Eluizione We can finish this part on HF FlFFF basics with something about efficiency. This expression for the equivalent plate height is general for FFF and , as in chromatography, it is obtained by the linear combination of different terms that contributes to peak broadening. As in all microcolumn separation methods, the the first critic contribution is relevant to the sample injection. In HF FlFFF we thus perform sample focusing before the elution, to make the sample plug as narrow as possible. Focusing is performed by injecting the sample with part of the mobile phase flow getting inside the channel from the outlet. The second point related to efficiency concerns the sample load. Sample load is critic in HF FlFFF because of the concentration effects of the sample band in the vicinity of the accumulation wall and because the reduced channel volume of HF FlFFF channels. In the very clever expression here reported and recently given by van Bruijnsvoort, Kok &Tijssen, we can see that the lower the limit of detection the higher the efficiency. The third point related to efficiency concerns the channel radius. We have shown in a previous slide that retention is dependent on channel radius. Here we see that the lower the radius, the higher the efficiency. So, it’s important not only to have the thinnest possible fiber, but also to keep the radius constant. van Bruijnsvoort, M.; Kok, W.Th; Tijssen, R. Anal Chem 2001, 73, 4736

23 Spettro ES/MS del lisozima separato in FFF dalla BSA
+8 +7 +9 Peso Molecolare misurato: ±1.05 Da Spettro ES/MS del lisozima separato in FFF dalla BSA

24 Spettro ES/MS della BSA separata in FFF dal lisozima
Peso Molecolare misurato: ±4.38 Da Liso+8 Liso+7 +40 +35 +30 +27 +43 Spettro ES/MS della BSA separata in FFF dal lisozima