Field-flow fractionation


                   Sample
                                          External field




        ...
AF4


Flow                Flow
 In                  Out
                                  Depletion wall
                 ...
AF4: working range


                               molecules            particles

       Atom         Molecule          ...
AF4: normal mode


                                                kT
                                     D=
            ...
Hyphenation



  AF4 retention depends on D and, then, on Mr
  AF4-based Mr measurements do not account for
  non-ideality...
Size/shape characterization by AF4-MALS



MALS     rg or RMS – mass average (root mean
square) distance of each point in ...
Light scattering properties




1. The amount of light scattered is directly proportional to the
   product of the molar m...
What does rg tell about our analyte?


                                                                  =∑
              ...
What’s the hydrodynamic radius?




            Rh         Rh                       Rh
       +
 _ H2O           H2O
     ...
rg / rh: a conformational index


                             3-arm star polymer

 solid sphere




                   Sa...
QDs: size-dependent fluorescence




                                 nm




        S. Kim et al. Nat. Biotechnol. 2004, ...
Biocompatible, water-soluble QDs



                        0.0   3.0    6.1     9.2     12.7   17.0   23.0      34.8
    ...
AF4-MALS-FD of water-soluble QDs
AF4-MALS-FD of fluorescent silica NPs




                             FLUORESCENCE FRACTOGRAM (λex: 325 nm; λem: 500 nm)
...
AF4-MALS-FD of fluorescent silica NPs



  Unbound
BLUE + GREEN                                      BLUE + GREEN NPs
    ...
Viruses




Denser inside




 courtesy from MedImmune Inc.
Virus-like NPs




courtesy of G. Winter et al. (2007)
Liposomes



                                             Liposomes or phospholipid vesicles emerged
                     ...
Liposome PSD: SEC-PCS vs. AF4-MALS




                            Egg phosphatidylcholine (egg PC, E-80)
                ...
Filled vs. unfilled liposomes




                         Radius versus elution time for a filled and unfilled liposome s...
Phospholipid nanovesicles for ophtalmic use


                                                                            ...
Layer-by-layer coated gold NPs for blood-blain barrier drug delivery




d=15 nm




                                     ...
AF4-MALS of multilayered gold NPs

                                                                                       ...
F4 for analysis of protein products




 F4 advantages

Wide Range of Applicability

Gentle Separation Mechanism

 Broad M...
Eclipse-DAWN HELEOS of BSA

                                                  molar mass vs. time/volume
                 ...
What makes protein drugs different?


    Protein drugs differ from low molecular weight drugs in terms of
    structure, ...
Protein aggregation: what’s the problem?




   Despite enormous technological advances made in the production and
formula...
Protein aggregation: analytical challenges


                    In spite of the enormous progress made in analytical
    ...
Protein aggregation is a method-dependent issue
                               We need the right toy…..
AF4-MALS of prion aggregates
β-Amyloid protein (Aβ) aggregation in AD


   The Aβ derives by secretase
   cleavage from the
   transmembran, amyloid
  ...
Aggregation of an amyloid peptide: Aβ1-42



                            UF                                               ...
Aβ 1-42 aggregation follow-up by AF4 and MALS/1
AE   1-42   aggregation follow-up by AF4 and MALS/2
AF4 of IgG

                      IgG pharmaceutical formulations in PBS

m AU



  50
                                  m...
AF4-MALS of Abs




High molar mass
     aggregates
Abs: IgG self-association /1




   Two different IgG samples show
same molar mass but different retention
Antibodies: IgG self-association /2




Zoom-in: dimer retention identical in both the IgG samples
            Only the mo...
IgG aggregation: „invisible“ and „visible“ particles




Fluorescence photomicrographs of Antibody A (from Novartis Pharma...
AF4-MALS of the IgG „invisible“ particles




Antibody A solution in 0.1% acetic acid containing 50 mM magnesium
chloride....
Pre-MS method for protein analysis
                                      ProsCons

                                       ...
F4 as pre-MS step for protein analysis



Broad application range
   High molecular weight proteins
   Protein complexes, ...
AF4 with nanoLC-ESI/MSMS for proteomics

                                                1.0




                      Rel...
Fraction collection from AF4


                                                                               LDL         ...
Molar mass distribution in the fractions




                                              8
Increasing retention time



...
Protein identification in VLDL fraction


                     1.0
Relative Intensity


                                  ...
Interactomic networks in VLDL fraction



   Fraction 8 8
   Fraction
                                    Dermcidin precur...
Hollow-fiber FlFFF (HF5)




   The HF5 cross-flow is generated by the elution flow, which splits
 into a longitudinal and...
HF5: prototype channel



                      1/8”
                    PE fitting


                                   1...
HF5: advantages




– Potentially disposable
  No risks of run-to-run sample carry-over
      – No memory effects when cou...
F4MS for protein analysis

                                                                                               ...
About byFlow R&D
About byFlow R&D
About byFlow R&D
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About byFlow R&D

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About byFlow R&D

  1. 1. Field-flow fractionation Sample External field th ad re B Thickness Flow External field Parabolic flow Thickness Detector v1 v3 v2
  2. 2. AF4 Flow Flow In Out Depletion wall (polymer, glass) Spacer UF membrane Frit (ceramic, metal) Cross flow Accumulation Out wall (polymer, metal)
  3. 3. AF4: working range molecules particles Atom Molecule Solids 1E0 1E2 1E4 1E6 1E8 1E10 1E12 Molar Mass 1 nm 10 nm 100 nm 1 µm Radius -Macromolecules: proteins, protein complexes, nucleic acids. -Nanopartcles: viruses, virus-like particles liposomes, lipoproteins, protein aggregates, subcellular components. -Microparticles: large protein aggregates, whole cells.
  4. 4. AF4: normal mode kT D= 6πη rh x Axial flow ⎛ xU x ⎞ C( x) = C0 exp⎜ − ⎜ D ⎟ ⎟ ⎝ ⎠ l z C Cross flow F = fU V ∝ 1/D ∝ rh r
  5. 5. Hyphenation AF4 retention depends on D and, then, on Mr AF4-based Mr measurements do not account for non-ideality effects D Mr relationship depends on analyte conformation Hyphenation with uncorrelated methods multiplies the amount of analytical information Hyphenation of AF4 with: Multi-Angle Laser Scattering Detection (MALS) Fluorescence Detection (FD) Mass spectrometry (MS)
  6. 6. Size/shape characterization by AF4-MALS MALS rg or RMS – mass average (root mean square) distance of each point in a molecule/NP from the molecule/NP center of gravity. 10nm – 1 P m AF4 rh or Hydrodynamic radius – radius of a sphere with the same diffusion coefficient of the analyte. 1nm – 100 P m
  7. 7. Light scattering properties 1. The amount of light scattered is directly proportional to the product of the molar mass and the sample concentration. [The amount of light scattered (divided by the incident light intensity) by a solution into a particular direction per unit solid angle in excess of the amount scattered by the pure solvent is directly proportional to the product of the weight-average molar mass and the concentration. R(θ), in the limit as θ→0, ∝Mc] 2. The variation of scattered light with scattering angle is proportional to the average size of the scattering molecules. [The variation of light scattered with respect to sin2θ/2, in the limit as θ→0, is directly proportional to the average molecular mean square radius. dR(θ)/dsin2θ/2 ∝ <rg2>]
  8. 8. What does rg tell about our analyte? =∑ ri 2 mi rg2 M hollow sphere: rg2 = a 2 solid sphere: rg2 = 5 a 2 3 L2 Random coil polymer with average end-to-end length L: r g 2 = 6
  9. 9. What’s the hydrodynamic radius? Rh Rh Rh + _ H2O H2O + H2O + H2O H2O
  10. 10. rg / rh: a conformational index 3-arm star polymer solid sphere Same rh rg rg ρ= = 0.77 ρ= ≈ 1.4 rh rh
  11. 11. QDs: size-dependent fluorescence nm S. Kim et al. Nat. Biotechnol. 2004, 22, 93
  12. 12. Biocompatible, water-soluble QDs 0.0 3.0 6.1 9.2 12.7 17.0 23.0 34.8 1000 1,0 15 nm Vis @ 533 nm Refractive Index Rayleigh Ratio 100 0,8 Relative Scale rms radius (nm) 0,6 3-5 nm 10 0,4 1 3-10nm ? 0,2 0,1 0,0 0,01 0 2 4 6 8 10 12 14 Retention time (min) (ca. 3 nm)
  13. 13. AF4-MALS-FD of water-soluble QDs
  14. 14. AF4-MALS-FD of fluorescent silica NPs FLUORESCENCE FRACTOGRAM (λex: 325 nm; λem: 500 nm) 3,5 100 90 3,0 80 2,5 70 rms radius (nm) 2,0 60 100 nm Fl (LU) 50 1,5 40 1,0 30 0,5 20 10 0,0 0 0 5 10 15 20 25 30 Time (min)
  15. 15. AF4-MALS-FD of fluorescent silica NPs Unbound BLUE + GREEN BLUE + GREEN NPs NPs NPs (497 nm) Unbound (463 nm) Elution Time (min) λex 325 nm BLUE > GREEN FRET AF4-FD gives direct evidence of FRET between the tandem dyes (BLUE > GREEN) This can be used for fine tuning of NP optical properties by changing the dyes ratio
  16. 16. Viruses Denser inside courtesy from MedImmune Inc.
  17. 17. Virus-like NPs courtesy of G. Winter et al. (2007)
  18. 18. Liposomes Liposomes or phospholipid vesicles emerged during the past 25 years as versatile and potent carriers for drugs and diagnostics, both, low-molecular weight compounds and peptide-/protein-drugs and genetic material. The size distribution of liposomal drug carriers is of key interest because size not only affects the vesicle’s in-vitro characteristics such as the amount of drug that can be accommodated, but also its in vivo behaviour such as circulation time in the blood-stream upon i.v.-injection, and consequently also biodistribution. Schematic drawing of small unilamellar l i p o s o m e - d r u g c a r r i e r Liposomes smaller than 70 nm can escape with hydrophilic drug in the aqueous through the fenestrae of the liver sinus.For compartments and lipophilic drug liposomes bigger than 200 nm, the steric incorporated into the phospholipid bilayer shield appears to be less efficient.
  19. 19. Liposome PSD: SEC-PCS vs. AF4-MALS Egg phosphatidylcholine (egg PC, E-80) liposomes by Lipoid GmbH, Germany The AF4-MALS approach has slight advantages being less time consuming, having lower preparative effort and thus shows less sources of error than SEC-PCS. However, AFMALS has limitations for very small liposomes. An additional online-coupling of PCS to the MALS detector might improve the detectability of very small liposomes. S. Hupfeld et al. J. Nanosci. Nanotechnol. 6, 1–7, 2006
  20. 20. Filled vs. unfilled liposomes Radius versus elution time for a filled and unfilled liposome sample 160 unfilled liposome filled liposome RMS- Radius (nm) 120 80 40 0 0 40 80 120 160 Elution time (min) courtesy from Wyatt Technology Europe GmbH
  21. 21. Phospholipid nanovesicles for ophtalmic use Effect of cholesterol uptake 1.2 2 0.5 mL LipimixTM detector voltage (V) 1.0 1 20 ng cholesterol 0.8 Effect of change in osmolarity 0.6 0.4 350 2.7 mOsm 10.0 20.0 30.0 40.0 50.0 60.0 27 mOsm time (min) 300 Root mean square radius (RMS, nm) 270 mOsm 1: native LipimixTM nanobeads 250 300 mOsm 2: higher-order structures 200 0.7 2 0.5 mL LipimixTM 150 200 ng cholesterol detector voltage (V) 0.6 100 1 50 0.5 2 0 0.4 0 5 10 15 20 25 30 35 40 45 50 Retention time (min) 10.0 20.0 30.0 40.0 50.0 60.0 time (min)
  22. 22. Layer-by-layer coated gold NPs for blood-blain barrier drug delivery d=15 nm G Schneider, G Decher Nano Letters (2006), 6, 530-536 Poly Allylamine Sodium Polystyrene Hydrochloride (PAH) Sulfonate (PSS) MW = 15 kDa MW = 4.3 kDa
  23. 23. AF4-MALS of multilayered gold NPs Au-PAH/PSS (Au core: 7.5 ±1.5 nm) 50 PSS NPs 45 40 35 [ Free polymer separated rms radius (nm) Signal Intensity 30 from the NPs 25 20 15 10 5 Au-PAH/PSS/PAH 0 80 0 2 4 6 8 10 12 14 Retention time (min) 70 UV signal @ 230 nm ( ), MALS signal @ 90° ( ) PAH 60 rms radius (nm) Signal Intensity 50 NPs 40 Higher state aggregation for triple-layer NPs 30 20 10 0 0 5 10 15 20 Retention time (min)
  24. 24. F4 for analysis of protein products F4 advantages Wide Range of Applicability Gentle Separation Mechanism Broad Mobile Phase Options F4 can be used to study high-MW protein products under native conditions and in formulation buffers
  25. 25. Eclipse-DAWN HELEOS of BSA molar mass vs. time/volume BSA 1mgmL 60uL 490um 3zu1 04[5Runs].vaf 4mer 3mer molar mass (g/mol) 2mer 1.0x10 5 1mer 20.0 25.0 30.0 35.0 time or volume FlFFF-UV fractogram and molar masses measured by on-line MALS
  26. 26. What makes protein drugs different? Protein drugs differ from low molecular weight drugs in terms of structure, source, analysis, formulation, and administration Protein drugs can undergo a variety of degradation reactions at the level of primary structure and higher-order structures The stability of a protein drug very much depends on how it is formulated Protein aggregates are an important class of degradation products that is difficult to tackle analytically and formulation-wise Aggregation can lead to the formation of soluble or insoluble aggregates, reversible or non-reversible aggregates, covalent or non-covalent aggregates Aggregates can vary in size from small dimers to large fibrils and be composed of native or misfolded protein molecules
  27. 27. Protein aggregation: what’s the problem? Despite enormous technological advances made in the production and formulation of protein drugs, the understanding, detection, and prevention of aggregate formation remain major pharmaceutical challenges Aggregates not only can have a reduced potency or show different pharmacokinetics, but also – even at extremely low aggregate levels – can cause serious safety problems Clinical implications of protein aggregates in a formulation are currently largely unpredictable and likely to depend on the aggregate species
  28. 28. Protein aggregation: analytical challenges In spite of the enormous progress made in analytical technologies to examine the chemical and physical integrity of protein aggregates, their full characterisation is not as yet possible A major complicating factor in the analysis of protein aggregates is that several aggregate types, in minute amounts, can coexist in one formulation, yielding a heterogeneous product The analytical challenges of studying early-stage, low levels of aggregated protein are huge: 1. it is difficult to pick up minor fractions of aggregated species in the presence of excess native protein 2. no single technique can detect all possible aggregates, so complementary techniques are necessary F4 or SEC combined with MALS are increasingly used to measure the physical properties of protein aggregates
  29. 29. Protein aggregation is a method-dependent issue We need the right toy…..
  30. 30. AF4-MALS of prion aggregates
  31. 31. β-Amyloid protein (Aβ) aggregation in AD The Aβ derives by secretase cleavage from the transmembran, amyloid precursor protein (APP) Three forms of Aβ: the Aβ1-42 peptide is most hydrophobic, most aggregating, and then most neurotoxic form of Aβ • Aβ1-42 is most lipophilic, and exists in two conformations: relaxed or a-strain bundled • Its size does not protect the inner lipophilic part from conformational changes • This originates self-assembling into oligomers, protofibrils, and insoluble fibrils
  32. 32. Aggregation of an amyloid peptide: Aβ1-42 UF Aβ 1-42 CE FILTRATED trimers-undecamers RETAINED dodecamers 50 kDa H y d r o d y n a m ic r a diu s (nm) 1 4 7 10 13 17 21 0,08 500 Rh ~ 5 nm L ~ 1.5 µm (AU) 0,06 MW ~ 60 kDa rms radius (nm) 400 Rayleigh Ratio 0,06 2 2 0 n m 300 0,04 t0 0,04 days 200 0,02 @ 0,02 100 A b s 0,00 0 0,00 0 5 10 15 20 25 30 0 2 4 6 8 10 Retention time (min) Retention time (min) AF4-UV AF4- MALS
  33. 33. Aβ 1-42 aggregation follow-up by AF4 and MALS/1
  34. 34. AE 1-42 aggregation follow-up by AF4 and MALS/2
  35. 35. AF4 of IgG IgG pharmaceutical formulations in PBS m AU 50 m AU ZOOM 3.5 3 40 2.5 2 1.5 1 30 Ab1 0.5 Ab2 0 -0.5 Ab3 -1 8 10 12 14 16 mi 20 Ab6 10 monomer dimer 0 8 10 12 14 16 m in MONOMER DIMER
  36. 36. AF4-MALS of Abs High molar mass aggregates
  37. 37. Abs: IgG self-association /1 Two different IgG samples show same molar mass but different retention
  38. 38. Antibodies: IgG self-association /2 Zoom-in: dimer retention identical in both the IgG samples Only the monomer self-associates
  39. 39. IgG aggregation: „invisible“ and „visible“ particles Fluorescence photomicrographs of Antibody A (from Novartis Pharma AG) dissolved in 0.1% acetic acid containing 50 mM magnesium chloride (A) and in 10 mM phosphate buffer pH 7.1 (B). No aggregates were visible in 0.1% acetic acid containing 50 mM magnesium chloride, even though protein concentration was high (94 mg/ml). Antibody A solution in phosphate buffer (0.8 mg/ml) showed many spherical aggregates, with a mean diameter of 3.18 μm B. Demeule et al. (2007) BBA 1774:146-153
  40. 40. AF4-MALS of the IgG „invisible“ particles Antibody A solution in 0.1% acetic acid containing 50 mM magnesium chloride. The monomer peak at 12.5 min shows a molecular weight of 170 kDa, whereas the aggregates peak at 18.5 min exhibits molecular weight ranging from 1 to 2 million Da. A magnified view reveals a smaller peak that can be a dimer.
  41. 41. Pre-MS method for protein analysis ProsCons 2D PAGE ↑Resolution, visualization, cost ↓Time, recovery, manual, off-line FFE ↑Unlimited throughput ↓pI-based selectivity only, ampholines CZE ↑Resolution, “nano-scale” ↓Saline buffers, on-line coupling issues RP LC ↑ Automated, desalting, easy on-line coupling ↓Sample denaturation, adsorption, recovery SEC ↑Size/shape-based selectivity ↓Sample entanglement Immunoaffinity ↑ Specific protein depletion ↓Co-depletion, desalting, cost Beads ↑Sample “equalization”, simple ↓Co-depletion, desalting
  42. 42. F4 as pre-MS step for protein analysis Broad application range High molecular weight proteins Protein complexes, aggregates, organelles Soft fractionation mechanism Biocompatible mobile phases Preservation of native conditions Evaluation of D Independent Mr determination MS-compatible mobile phases No ionization suppression
  43. 43. AF4 with nanoLC-ESI/MSMS for proteomics 1.0 Relative Rayleigh ratio 0.8 0.6 0.4 0.2 0.0 0 20 40 60 80 Retention time (min) Enzymatic hydrolysis (trypsin) nanoLC – ESI/MSMS
  44. 44. Fraction collection from AF4 LDL VLDL 1.0 7 8 UV (280 nm) Relative Intensity 0.8 Rayleigh Ratio 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Retention time (min) 1.0 113000 80000 Relative Intensity 0.8 HDL + 51800 0.6 HAP 34700 30000 0.4 22000 0.2 0.0 0 1 2 3 4 5 6 7 8 Retention time (min) SDS-PAGE
  45. 45. Molar mass distribution in the fractions 8 Increasing retention time 7 Fraction number 6 Lower-Mr 5 components found in the 4 fractions of 3 higher-Mr components 2 1 0 10 100 1000 10000 Molar mass (KDa)
  46. 46. Protein identification in VLDL fraction 1.0 Relative Intensity 8 8 UV (280 nm) 0.8 Rayleigh Ratio 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Retention time (min) NAME ACC NUM SCORE MASS Serum albumin precursor P02768 1178 71317 Keratin, type II cytoskeletal 1 P04264 800 66018 Complement C3 precursor P01024 281 188585 Keratin, type I cytoskeletal 10 P13645 268 59711 Keratin, type II cytoskeletal 2 epidermal P35908 265 66111 Ig kappa chain C region P01834 255 11773 Dermcidin precursor P81605 221 11391 Alpha-2-macroglobulin precursor P01023 194 164600 Transthyretin precursor P02766 191 15991 Ig gamma-1 chain C region P01857 180 36596 Prothrombin precursor P00734 170 71475 Alpha-1-antitrypsin precursor P01009 153 46878 Apolipoprotein C-II precursor P02655 148 11277 AMBP protein precursor P02760 137 39886 Apolipoprotein A-II precursor P02652 136 11282 Keratin, type II cytoskeletal 6A P02538 123 60162 Ig lambda chain C regions P01842 119 11401 Alpha-1-acid glycoprotein 1 precursor P02763 108 23725 Haptoglobin precursor P00738 100 45861 Apolipoprotein A-I precursor P02647 98 30759 Inter-alpha-trypsin inhibitor heavy chain H4 precursorQ14624 96 103489 Ig kappa chain V-II region Cum P01614 93 12782 Protein S100-A7 P31151 91 11433 Beta-2-glycoprotein 1 precursor (Apolipoprotein H) P02749 89 39584 Apolipoprotein C-III precursor P02656 85 10846 Ceruloplasmin precursor P00450 85 122983 Alpha-1-antichymotrypsin precursor P01011 64 47792 Apolipoprotein E precursor P02649 63 36246 Nuclear mitotic apparatus protein 1 Q14980 41 239214 Myeloid/lymphoid or mixed-lineage leukemia protein O14686 40 570046 Ig alpha-1 chain C region P01876 38 38486 Abnormal spindle-like microcephaly-associated prote Q8IZT6 37 413192 Immunoglobulin J chain P01591 36 16041 Development and differentiation-enhancing factor 2 O43150 35 112835 Sodium/potassium-transporting ATPase alpha-2 cha P50993 32 113505 Apolipoprotein C-I precursor P02654 32 9326 Calmodulin-like protein 5 Q9NZT1 31 15911
  47. 47. Interactomic networks in VLDL fraction Fraction 8 8 Fraction Dermcidin precursor Dermcidin precursor Prothrombin precursor Prothrombin precursor Apolipoprotein A-II precursor Apolipoprotein A-II precursor Keratin, ,type IIII cytoskeletal6A Keratin, type cytoskeletal 6A Transthyretin precursor Transthyretin precursor Apolipoprotein C-I precursor C-I precursor Apolipoprotein Keratin, type II cytoskeletal 1 Keratin, type II cytoskeletal 1 Apolipoprotein A-I precursor Apolipoproteinprecursor A-I HSA precursor Serum albumin precursor Apolipoprotein C-III Apolipoprotein precursor C-III precursor Igalpha chainC region -1 Haptoglobin precursor Haptoglobin precursor Keratin, type II cytoskeletal 10 Keratin, type cytoskeletal 10 IgIg gamma-1 chain C region gamma-1 chain C region Ig kappa chain C region Ig kappa chain C region Ig lambda chain C regions Apolipoprotein C-IIprecursor C-IIprecursor Apolipoprotein
  48. 48. Hollow-fiber FlFFF (HF5) The HF5 cross-flow is generated by the elution flow, which splits into a longitudinal and a radial direction: no depletion wall, only accumulation wall Cross-flow outlet Hollow Fiber r rf Channel Vin z Vout sleeve Cross-flow Inlet connection Tee connection Outlet connection (from injector) (to detector)
  49. 49. HF5: prototype channel 1/8” PE fitting 1/8” 1/8” 1/8” PEEK Tee Teflon tube cPVC / PSf 1/8” 1/8” HF membrane PEEK ferrule PEEK ferrule 24x0.08 ID cm PEEK Nut (1/8”) Hollow Fiber Ferrule (1/8”) Teflon sleeve Union Tee (1/8”)
  50. 50. HF5: advantages – Potentially disposable No risks of run-to-run sample carry-over – No memory effects when coupled with other techniques Reduced sterility issues – Easier work with biological samples – Low channel volume Low sample dilution – High detection sensitivity Short analysis time – Highly suitable to hyphenation
  51. 51. F4MS for protein analysis HF5 of BSA: 1 fractionation of oligomers 1: monomer 2: dimer 3: trimer 4: tetramer 2 3 4 +50 +45 100 100 M = 66397.8 +55 +40 (monomer mass) % % +36 +60 0 mass mass 56000 58000 60000 62000 64000 66000 68000 70000 72000 74000 76000 78000 80000 0 m/z 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 ESI MS multicharge spectrum ESI MS mass spectrum

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