Importance of sample preparation
S2004
Methods for characterization of biomolecular interactions
- classical versus modern
Mgr. Josef Houser, Ph.D. houser@m ail. muni, cz
Ideal sample - ideal data
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Time (min)
■ D120092016 NPH -D12009201ö_B
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Chr2/DoF = 20BS
N 0 931   ±0.00373 SteE
K 4.92E4 ±1.S3E3 H~'
}H       -6200   ±29 00ca^mo
nS       6 675 cttnolftlea
0 0     0 5      10     15     2 0     2 5     3 0 Malar Ratio
C
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Time/ seconds 10 20 30 40
1.05
1.00
0.95
if 0.90
0.85
0.80
0.75
1000 950 900 850-800-750-
KD = 75.3±2.01 uM
10-2   10-1   10°   101    102 103 [25]/ uM
104 105
Real data - not always that ideal
Tim e (m in)
0     10   20   30   W   SO   60   70   SO   90 100
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MST experiment time [s]
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+2                                    +3 +4 1x10                           1x10                           1x10 1x1 Ligand Concentration																				+ 5 +6 0 1x10					
Reproducibility crisis
• Based on 2016 poll with > 1500 scientists included:
70 % were not able to repeat an experiment!
50 % were not able to repeat at least one of their own experiments !!!
• Possible causes:
• Selective choice of data (cherry picking)
• Unsuitable experimental desing
• Inappropriate data evalueation (statistics)
• It's probable that partial problem is insufficient characterization of input material and procedures.
Source: nature.com
4
Ideal sample properties
• Defined (chemically, biologically, conformationally)
• Pure (contamination by small molecules, macromolecules)
• Homogeneous (micro-/rriacro- heterogeneity)
• Stable (storage, time-demanding analysis)
Sample identity
Exact composition of sample (sequence, modifications, cleavage) Influence on MW, pi, interactions
Covalent oligomerization
Glycosylation
iaHIA99^DAISNSN9AA^DSHAAIISiaN9ANAIANANSAiaSSS¥9AaSJ
iswiAAasaATsaaiiiaaiiNNDANsDaasaAassaaisaisaAYSwaYDSj
¥I¥iaDASDlA9aiAAI¥NINa9¥aAlDaWI9SIH¥S¥ISATS¥¥WISnaD>
Signal peptide )(
1MQFLTSLAAAASLVSLASAftlSGIALPQTVKAGDNINAIVVTEGYIQSVQDIAIAFGCAPAA
SAYPGTLSTLLGSFYLGPEQCNVQNNITEPITIPESLVPGEYVIAASLFSLYGASSSPTVSN
I
JYNVTVNVGNET^tCTYVRSQFCVGNSNSTVGLGGYTRKINALSGTVA PO4
Degradation Phosphorylation
Methylation
Sample identity
• MS identification
1 MKKESINTSG PDNTKSSISD
51 GVGTNNAVWH NWQTVPNTGS
101 RGTDNALWHN WQTVPGAGWS
151 DNALWHIWQT APHAGPWSNW
201  SLWYIKQTAS HTYPWTNWQS
251 WHIWQVAPNA GWTNWRSLSG
301 WQTATSDAWS EWTSLSGVTT
351 TSSWSTWTSL GGNLIDASAI
EIEISNEISW TALSGVISAA NNADGRLEVF
SWSGWHSLNE GATSKPAVHI NSDGRLEVFV
GWQSLGGQIT SNPWYINSD GRLEVFARGA
QSLNGVLTSD PTVYVNASGR PEVFARSNDY
LSGVITSNPV VISNSDGRLE VFARGSDNAL
IITSDPAVHI NADGRLEVFA RGPDNALWHI
SAPTVAKNSD GWLEVFARGA NNALCHIQQT K
MS intact mass analysis
abundance				
4000				
3000				
2000				
				40462
i (unlit.		[MHj]3'		L
1U00O
20000
30000
40000
50000
60000
Post-translational modifications Isotope labeling Matrix adducts
m/z
Folding - direct evidence of 2D structure
* Circular dichroism (CD)
• Difference in absorption of left and right circularly polarized light by chiral compounds
• Specific shape of spectra for 2D structural elements
* Infrared spectroscopy (FTIR)
• Specific absorption bands for 2D elements
* Nuclear magnetic resonance (NMR)
• Behavior of atom nuclei in magnetic field
• Presence of defined structure results in distinguished peaks in spectrum
200 210
220 230        240 250
Wavelength (nmj
Dodero 2011
Unfolded			Folded ■ ,	a
			- 4**	
				
				t
				
* •			0	
9.0      8.0 7.0 1H Chemical shift (ppm)
9.0      8.0 7.0 1H Chemical shift (ppm)
100 105
110 &
115 1 u
120 I to
125 z in
130 135
Balbach 1996
Sample purity
Contaminants - co-purified molecules • Small molecules
• Co-factors
• Ligands
• Salts, imidazole
• Lipids Saccharides
Macromolecules
• Protein isoforms
• Proteins
• Nucleic acids
• Polysaccharides
• Binding partners
Sample purity - methods
SDS-PAGE
UV-VIS spectroscopy SEC (SEC-MALS) FFF (FFF-MALS) Mass spectrometry
small molecules Co-factors Ligands
Salts, imidazole Lipids
Saccharides
macromolecules Protein isoforms
Proteins
Nucleic acids Polysaccharides Binding partners
10
SDS-PAGE
Polyacrylamide gel (8 - 20 %)
SDS - uniform (?) protein charge (composition dependent)
Reducing agent (optional) - (3ME
Staining - CBB, Silver, Fluorescent, Radiological
l-
Sensitivity
Coomassie staining
5-25 ng
Silver staining
0 25-0.5 ng
0.25-0 5 ng
11
SDS-PAGE
• Check overloaded as well as underloaded sample
UV-VIS spectroscopy
(200-) 240-340 nm
• Trp (and Tyr) has absorption peak around 280 nm
• Detection of:
• Nucleic acid contamination
• Aggregation (scattering)
• UV-absorbing contaminants
Nucleic acids
arbre-mobieu.eu
260 280 300
Wavelength nm
Scattering
340 2*0 796  500  120 J40
Wavelength nm
	OS -
*J	04
1	05 •
1	02-
1	
	01 •
	0 ■
320
340
13
Size exclusion chromatography
• Separation of particles based on "size"
• Interaction with matrix possible (!)
• Usually coupled to multiple detectors
(UV, MALS, viscosity)
2.0 E+05
5 10 15 20
Elution volume (ml)
Field flow fractionation
• Separation of particles in solution by external force
Field Flow Fractionation
Outlet to the detector
Separation field
Parabolic flow profile Diffusion
Separation field
Field of force
Techniques
r
Thermal
ThFFF
-1-
Centrifugal
Flow I
T
Electrical
SdFFFor CFFF
1
Magnetical
FIFFF (SF4)	EIFFF		MgFFF
AF4	CEIFFF		
HF5			
Mass spectrometry
• Detecting of exact mass of particles
• Various applications based on set-up
• Intact mass analysis - protein and non-protein contaminants
Sample homogeneity
• Macroscopic - precipitation - visual detection
• Microscopic - oligomeric states, folding states, microheterogeneity - biophysical methods
Sample homogeneity vs. purity
Various methods may evaluate sample in different way
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Homogenous
Good sample
Sample homogeneity - methods
• SEC-MALS, FFF
• Native electrophoresis
• Light scattering
• Analytical ultracentrifuge
Native electrophoresis
• Possibility to observe various oligomers (relatively imprecise and unreliable) and isoforms (2D PAGE preferred)
• Not efficient for aggregation detection
Light scattering
• Interaction of incident light with particles in solution
• Intensity of light at given
• Typically red/infrared light
Detector
Light scattering
• Dynamic light scattering
- size of particles
- sensitive to aggregation
(a)
(b)
C(q.r)
• Static light scattering
- mass of particles
- averaged value, separation required
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arbre-mobieu.eu
Large Particles
oo
Small Particles
o o oo o
1(11)
Log(r)
Log(ri
Static light scattering (SLS)
Low-angle light scattering (LALS) - big molecules Right-angle light scattering (RALS) - small molecules
Multi-angle light scattering (MALS) - Mw and Rg
• Intensity of scattered light
• Mass of the particle (molecular weight)
www.malvernpanalytical.com
Static light scattering
• Average of all sample particles !
• Typically coupled to separation (SEC, FFF)
Retention Time (mm]
volume {ml_)
Dynamic light scattering (DLS)
Time-dependent fluctuations in scattered light Size of the particle (hydrodynamic radius)
Photon Counting Detector
Autocorrelator
Allen P Minton,
DOI: 10.1016/j.ab.2016.02.007
(a)
m.0
(b)
C(q,r)
lt(q.0)]'~
Large Particles
oo
Small Particles
o o
Oo o
I(q.l)
C(q,r)
Log(r)
Log(r)
Dynamic light scattering (DLS)
Shape dependent Low resolution
For ideal sphere:
•
M ~ V = 4/3 ti r3
M2 = 2 X M1
r2 = V2 x r± = 1.26 r±
rJdimer) ~ 2 X rjmonomer)       rh(dimer) ~ rh(monomer)
Dynamic light scattering (DLS)
- Microheterogeneity reflects in polydispersity - peak width
- Large particles scatter light with much higher intensity - sensitive to aggregation
0.01 1 102        104 0.01 1 102 104
Radius (nm) Radius (nm)
27
Analytical ultracentrifugation (AUC)
• Sedimentation of particles in centrifugal field by hydrodynamic properties
• Two modes:
• Sedimentation equilibrium - mass determination
• Sedimentation velocity - size distribution
monodisperse sample polydisperse sample
sedimentation coefficient (S) sed i me ntatio n co effic i e nt (S)
Comparison
	Light scattering	Analytical ultracentrifugation
Sample volume	0.5-30 ul (DLS) 1-50 ul (SLS, SEC-MALS)	150-450 ul
Sample concentration	0.1-200 mg/ml	0.1-1 mg/ml
Particle size	1 nm - 10 pirn	1-300 nm
Resolution and accuracy	Low - Average	Average - High
Speed of analysis	1 min (DLS, SLS) 30 mins (SEC-MALS)	4 hrs (SV) 3-4 days (SE)
Sample stability
• Temperature stability
• Chemical stability
• pH
• Ionic strength
• Oxidizing agents
• Protein-specific compounds
• Long-term stability - storage
Temperature
• Affects stability and interaction parameters
• Typical temperatures:
-80 °C, -20 °C, 4 °C, 20 °C, 25 °C, 37 °C
• Room temperature (RT) - vaguely defined
mostly 20 - 25 °C, but varies from 15 - 30 °C usually means that temperature was not set (!)
AG
Arrhenius equation
31
pH=-log[H30+]
Typical range: 4-9, specific proteins 1-12
pH of pure water: 7 (theor.), 5.8 (due C02 absorption)
Buffers: dissociable compounds with defined pKa
various pH ranges - typically (pKa-l) - (pKa+l) ^
buffers
Organic/Inorganic
Universal buffers -mixtures with broad pH range
Good's Buffer	pKa (20 °C)	PH
MES	6.15	5.5-7.0
Bis-Tris	6.46	5.7-7.3
ADA	6.60	5.8-7.4
PIPES	6.80	6.1-7.5
ACES	6.90	6.0-7.5
MOPSO	6.95	6.2-7.4
BES	7.15	6.6-8.0
MOPS	7.20	6.5-7.9
TES	7.50	6.8-8.2
HEPES	7.55	6.8-8.2
TAPSO	7.70	7.0-8.2
POPSO	7.85	7.2-8.5
HEPPSO	7.90	7.4-8.6
EPPS	8.00	7.5-8.5
Tricine	8.15	7.8-8.8
Bicine	8.35	7.7-9.1
TAPS	8.40	7.7-9.1
CHES	9.50	8.6-10.0
CAPS	10.40	9.7-11.1
a i m s Biophysics
hup://www.aimspress.com/
Volume 2. Issue 3, 336-342. DOI: 10 3934/biophy.2015.3J36 Received date 19 April 2015. Accepted dale 20 July 2015. Published date 14 August 2015
Universal buffers for use in biochemistry and biophysical experiments
Dewey Brooke , Navid Movahed , and Brian Bothner *
Department of Chemistry and Biochemistry. Montana State University. Bozeman MT 59717. USA
PH
• is temperature dependent
• changes with dilution changes in time 81
Tr's buffer pH set to o n
90P ~~^Zzet to 8-0 at 25°r
— 6.4-        '   ™ ~ ■6.6" -6.8-
7.7 75
,afew^|o,0mo(/i;
Hressure [MPa]
~«» 1000
PBS* W*0      PRC „.
34
Ionic strength
Ionic strength, /, is a measure of the concentration of electrically charged species in solution
i
1.0 2.0 Square root of ionic sirenglu
Protein solubility changes with ionic strength as well as with solute composition
35
purities/Additives
OH
Various compounds affect protein stability/solubility ho^^o^o^J^dh
H    ^r^'"0 °^'"OH 0H  H ^OH
Saccharides - saccharose, trehalose Amino acids - Arg, Glu, Pro nh o
Reducing/oxidizing agents - pME, DTT, TCEP h2m^n-^^^
nh2
DMSO
Protein-specific compounds (ligands) 0 ,v.
OH
O OH HO^^P^^OH
C O
H3C CH3
36
Effect of impurities on ITC
Time (min)
0    10   20   30   40   50   60   70   80   90 100
Time (min)
0.5
Molar Ratio
0    10   20   30   40   50   60   70   30   90 100
I.
0.5 1 .□
Molar Ratio
o E
u
1.0
Molar Ratio
37
Effect of impurities on ITC - DMSO
10.00
0.00
u
J" -10.00 TO
u
-20.00
-30.00
0.00 20.00
]A231212201F -A231212201E
r
40.00 60.00
Time (min)
80.00 100.00
3.33
1)
i—m
i-1-1—
0.00 20.00
3M2131220K - M2131220K
'S 33 SO.0 0 S3 3 3 13 3 33
Timo /mini
DMSO buffer mismatch
0.5 |jcal/sec
m       ruin ŕ ^      ^      ^       E       t       Ŕ       A       A       k       h k,
nrrTTTTTTTTTTTTTTTT
IÍTTfíTíTÍ^^
XO]      III     [HHJ]    [l5ľo|    ^0ľÔ|    Í25ľÔ|    |30ľf3|    [Šitôl ^
Time (min)
Buffer into buffer 5% DMSO into 5% DMSO 5% DMSO into 4.5% DMSO 5% DMSO into 4% DMSO
B
B
5 mM ligand in 100% DMSO
\50 |al 950
Dialysate buffer
B
250 u.M ligand in 5% DMSO
0
DMSO
25 u.M dialýz protein
1 ml of 23.75 U.M protein in 5% DMSO
39
DMSO buffer mismatch
• Determine DMSO concentration
• Match for protein and ligand
B
Dialysate buffer
0
DMSO
V
250 nM ligand in "5%" DMSO In the syringe
1
Dialysate buffer 4.8% DMSO
Dialysate buffer 4.9% DMSO
Dialysate buffer 5.0% DMSO
Dialysate buffer 5.1% DMSO
Dialysate buffer 5.2% DMSO
77
W
Buffer optimization
• Buffer affects:
• Stability
• Activity (interactions)
• Storage
• Many buffers do not meet all requirements
Buffer optimization desired
Buffer optimization
Various commercial screens available
Differences in composition, number of conditions
Example: buffer screen designed by CF BIC, CEITEC MU
2 3
5 6
8      9     10     11 12
H20
pH 2-12
pH 4-9.5 (alternate buffers from A row)
Ionic strength (for pH 6-8)
Pre-defined buffers
Additives
42
Buffer optimization
	n	2	3	4	5	6	7	8	9 10	11	
	59.2°C		43.6°C	37.7°C	55.0°C 61.3°C		59.8°C	62.1°C	55.5°C 59.0°C	33.4°C 33.2°C	
B	36.5°C 42.1°C		48.3°C	52.2°C	55.0°C 58.5°C		66.2°C	66.4°C 66.5°C	58.7°C 59.4°C	63.1°C 63.3°C	
C	57.2°C 59.2°C 60.6°C 58.5°C		62.7°C	62.1°C	67.0°C 68.1°C		69.9°C		60.2°C 61.8°C	66.5°C	70.0°C
D			69.4°C	63.4°C 46.2°C		55.2°C   58.2°C 54.5°C			59.2°C 59.5°C	-	59.2°C
Buffer screen C7 + C12 condition
-1-1-1-1-1-1-1-
20 30 40 50 60 70 80 90 100
Temperature PC)
Sample storage
• Depends on sample stability
• Freezing (phase transition) may decrease protein stability in solution
Avoid repeated freeze-thaw cycles !
• Fridge: 4 °C
• Freezer: - 20 °C, - 80 °C (cryo-protectants addition - glycerol)
• Lyophylization = Freeze-drying: water sublimation
Check sample quality BEFORE and AFTER storage !
Practical aspects
Concentration
Method	+	—
Nitrogen content (e.g. Kjeldahl)	Absolute (golden standard)	Time, sample and equipment demanding
UV absorbance at 280 nm	Fast, easy, low sample consumption, no calibration	Sequence dependent, buffer influence, (inaccuracy in 1, e)
Bradford (Coomassie Brilliant Blue)	Easy, fast	Standard dependent (calibration), sequence dependent, buffer influence
Bicinchoninic acid	Less buffer dependent	Standard dependent (calibration), more time demanding
UV absorbance at 205 nm	Less sequence dependent, + the same as A280	Buffer absorbance
Pipetting
Pipetting is a science Many variables
• Viscosity
• Type of tip/pipette
• Immersion depth
• Angle
• Tip-pipette match
• Pipette holding
• Moisture
Raw Data Inspection
Capillary "icsi     CiQi^'y Shape     In its' Fluorescence     Bleaching Rate ©
iw Data Inspectior
Properties IvlST Traces Capillary Scan Capillary Shape Initial Fluorescence Bleaching Rate © F^l Zoom Extent        Export v
£ 100
7 8 9 10 11 12 13 14 15 16
Caoillarv Position f-1
Sample aging
• Check storage conditions
• Avoid freeze-thawing cycles as much as possible
• Check buffer pH - use freshly prepared buffers
• Batch-to-batch verification
48
Batch to batch quality check
• Enormous amount of variables in preparation process
• Two sample batches may not be the same
• Minimal tests desired to verify sample quality
Temperature
• Many machines keep specific temperature
• Check settings
• Room temperature varies over day, week, seasons
• Avoid "bad spots" in lab - heating, direct sun, air conditioning
| cs>CelPress Med Clinical and Translational Report
Variation in common laboratory test results caused by ambient temperature
Ziad Obermeyer1-3'4'" and Devin Pope?"i
L---m-
Methods are not identical
• Results from different methods usually vary
• Ideal match of values (e.g. Kd) is unlikely
• Some methods require specific sample preparation and conditions
• Know method principles and limitations !!!
• Know your sample !!!
Biomolecular I nteraction and rystallization Core Facility
Josef Houser
I •   +420 549 492 527
• josef.houser@ceitec.cz
bic@ceitec.cz bic.ceitec.cz
MUNI
CF Head: Michaela Wimmerovä
•   +420 549 498 166 michaela.wimmerova@ceitec.cz