Non-Naturally occuring GFP Analogs
Obtained through Mutagenesis / Synthetic modification
Diversity covers nearly the entire visible spectrum
Serius / BFP / CFP / YFT etc
Conjugation system –> FLUORESENCE
small changes that affect the conjugation –> various colors
FP = Fluoresence Proteins
in PROTEIN LABELING experiments
FP –> either C-terminus or the N-terminus of the target protein
sometimes BOTH are needed for function so:
FP -> inserted inside the protein chain
Gly-linker, common but not mandatory
offers flexibility/avoids steric conflicts
- *Expression levels MUST be monitored** to ensure that the protein
- *Still functions normally!**
Uses of GFP-type Imaging Experiments
HeLa Cells
multicolor labeling of the cells via confocal microscopy
Show where EACH PROTEIN is LOCALIZED
mitochondria / actin / histone / golgi / etc
Uses of IN VIVO Labeling using FPs
help visualize cell types in
whole animals / organs / tissues / cell cultures
Neurobiology / immunology / development
Transplantology / Carcinogenesis
Real-Time Monitoring of CELL CYCLE PROGRESSION
of living tissues
G1 Phase / S /G2 / M phases
LIMITATIONS of IN VIVO labeling using FPs
LIGHT ABSORPTION BY:
MELANIN
HEMOGLOBIN
has extensive conjugation –> absorbs some light
Optimal optical window for living tissues is:
650-700nm & 1,100nm
which is the “near IR” region = 700->1,700nm
Uses for NEAR IR PROBES
“NIR”
Visualization of ANATOMICAL FEATURES
image guided surgical removal of disease tissues
Blood+lymph vessels / GI tract / Bile Duct / Uterus
- *Biomedical Imaging**
- *high imaging resolution** with increasing tissue penetration depths
NIR-1 = 700-900nm & NIR-2 = 1,000-1,700nm
probes allow imaging of inorganic/organic macromolecules
Give 2 Reasons why FLUORESCENT PROBES are USEFUL
- *FPs encoded in proteins of interest** make it possible to observe their:
- *LOCALIZATION / MOVEMENT / TURNOVER**
- *FPs targeted to cells** enable visualization of:
- *MORPHOLOGY / MOVEMENT**
useful for studying disease states
What light spectrum does NMR measure?
RADIO WAVES
measure the
NUCLEAR SPIN
NMR Signals are the result of what?
Absorption of ELECTROMAGNETIC RADIATION (resonance)
by NUCLEI
NMR Steps
- Spectrometer
- gives of an electromagnetic pulse
- the sample absorbs this energy
- FID = Free Induction Decay
- electromagnetic radiation emission by each excited nuclei
- Fourier Transform
- digests the data / MATH
- SPECTRUM
- Representaion of nuclei in the molecule
- each nuclei emitted radiation at a DIFFERENT FREQUENCY
NMR Specrum is what?
Representation of the NUCLEI in the molecule
Each nucleus emitted radiation at a
Slightly different FREQUENCY
What can we measure and NOT measure with NMR?
NMR = Radiowaves that measure NUCLEAR SPIN
only a select few nuclei are good canidates for NMR:
NMR Nuclei
1H (99.9%), 13C (1.1%)
14N, 17O, 19F
elements that _LACK A NUCLEAR SPIN_ will _NEVER exhibit an NMR phenomenon_
What type of SOLVENTS do we use for NMR? and WHY?
DEUTERATED SOLVENTS
CD3OD / CDCl3
Deuterates = like 2H
are INVISIBILE in NMR experiments,
does NOT interfere with the signal
Our goal is to have:
minimal interference from the solvent
Differentiating SIGNAL in NMR
Nuclei have DIFFERENT electronic environments
Emit a DIFFERENT TYPE OF ENERGY
As a result these nucleu have resonance @ different FREQUENCIES
= X-axis of spectrum
- *Electrons** (electron density) SHIELD each nucleus from the
- *Applied Magnetic Field**
Downfield = ?
DE-Shielded
Further LEFT,
on the X-Axis (chemical shift or frequency, PPM or Hz)
GREATER Frequency
Upfield = ?
SHIELDED
Further RIGHT–>,
on the X-Axis (chemical shift or frequency, PPM or Hz)
lower Frequency
Chemical Shift (δH) INCREASES As……
ELECTRONEGATIVITY of adjacent atoms INCREASES
Chemical shift δH = position on the x-axis of the spectrum
protons in different electronic environments exhibit
Different Chemical Shifts
Chemical Shift δH
Value of a RESONANCE on the X-Axis of the Spectrum
Each electron environment causes a characteristic δH
which allows for us to predict the nature of the environment/type of the signal of interest (proton)
δH INCREASES as ELECTRONEGATIVITY of adjacent atoms INCREASE
Why is a hydrogen adjacent to an oxygen is more deshielded vs a hydrogen next to a carbon?
OXYGEN IS MORE
ELECTRONEGATIVE
The hydrogen (proton) is being “deshielded” by the Oxygen which is pulling electrons away
Functional groups such as -OH de-shield protons
Why is the CH2 adjacent to the ARYL GROUP
more SHIELDED
than a normal CH2 group?
the ARYL Group is a
CONJUGATED SYSTEM / AROMATIC GROUP
that SHIELDS the proton
The hydrogen (proton) is being shielded by the conjugated system which is contributing to the degree of electrons round the proton
What is INTEGRATION?
in terms of Spectral Analysis
The AREA UNDERNEATH A PEAK in a NMR spectrum
correlates directly with the number of nuclei involved
When is H NMR NOT HELPFUL in determining structures?
ABSENCE OF HYDROGEN ATOMS
a lot of CONJUGATION –> have less hydrogen atoms
lead to blind spots in the structure
so we would then use C-NMR
Negatives to 13C NMR experiments
+ Positives
13C is only 1.1% in relative abundance, not-abundant
LONGER EXPERIMENT TIMES
H1 is much faster because there is much more of them 99+%
- *13C NMR** has a LARGER range for X-axis Chemical Shifts
- *δC: 0 – 220 ppm**
appear as singlets
Types of NMR Methods
COSY H-H
NOESY / TOCSY = 2D H-H
2D Method = Box Plot
HSQC = H-C
HMBC = H-C
COSY
NMR Method
- *H-H**
- *COrrelation SpectroscopY**
HOMOnuclear Spin Coupling
Corralates hydrogens next to hydrogens.
Off-diagonal contours indicate coupling
proton-proton coupling of H-Groups
SHOW BONDS / DIRECT CONNECTIVITY
What NMR Method is useful in determining
Similar H-Groups next to the “same” H-group?
Ex. CH2-CH2 or CH=CH
COSY
H-H
Proton-Proton Coupling of adjacent H-Groups
HSQC
NMR Method
Heteronuclear Single Quantum Coherence
DIRECT H-13C heteronuclear spin coupling
Will ONLY show H’s that connect to C’s = CONTOUR
H on X axis
C on Y axis
matching will show that those two are connected
does not show quartenary carbons, or resonances due to -OH or -NH
Which NMR Method tells us
Which H is directly connected to which C?
HSQC
“H->C”
DIRECT heteronuclear spin coupling
HMBC
NMR method
- *H**eteronuclear Multiple Bond Correlation
- *H-C** Heteronuclear spin coupling
- like HCQC but shows MORE INFO*
2-3 Bond Correlations Observed
Correlation BETWEEN each structure
key to PIECING the structural fragments together
typically used after the other tests
Which NMR method shows us
the correlation BETWEEN each structure?
observes 2-3 bond correlations
HMBC
H-C
Shows more info than HCQC, typically used after the other tests
NOESY
NMR method
Nuclear Overhauser Enhancement SpectroscopY
H-H homonuclear spin coupling
DIPOLE DIPOLE THROUGH-SPACE INTERACTIONS
does NOT read through bond interactions
MAX 5Angstrom distance
Shows where things are pointing in space + Shape
Stereochemistry
Cis vs Trans
E vs Z
Which NMR method shows us
Stereochemistry (shape) of the sample and (stereo)Configuration of double bonds?
Cis / Trans
E / Z
NOESY
H-H
shows THROUGH SPACE PROXIMITY
MRI Facts
Magnetic Resonance Imaging
- *Same principles as NMR,**
- except we use the WHOLE BODY (Tube)*
We DONT simply “image” the whole body at once
signal would be too messy, must be fine tuned
Superconducting MAGNET
data collection produce high quality images
Mass Spectrometry Steps
- Sample IONIZATION
- shoots ENERGY
- Sample Breaks apart / separation
- forms FRAGMENTS
- DETECTION of these ions & fragments
degrative process
“Reverse Engineering”
Mass Spectrometry Definition
Any technique to achieve the introduction of a sample
followed by Ionization -> Seperation -> detection of ions
Destructive method,
allows you to observe how a metabolite fragments
High Resolution MS
Allows you to determine MOLECULAR WEIGHT
down to MORE decimals so that we can determine
More EXACT Molecular Formula
Molecular Ion Definition
MS
The ION obtained from the
LOSS of an electron
from a molecule M+
Base Peak Definition
MS
the MOST INTENSE peak in the MS
Fragment Ions Definition
MS
LIGHTER IONS fromed by the
decomposition of the molecular ion
Pseudo molecular ions, or adducts
MS
[M+H]+,
[M+Na]+,
[M-H]-, etc.
Obtaining STRUCTURAL INFORMATION from MS
GRAPH, what is Y & X?
Y = Relative Abundance
X = m/z , mass to charge ratio of fragment
SIMPLY A MATH PROBLEM
To identify fragment ions in a spectrum, determine the difference between the
[m/z value of a given fragment ion] - [that of the molecular ion]
Mass Spectrometry what bonds break and in what preference?
WEAK BONDS > strong bonds
weak bonds break more easily
Bonds that break to form MORE STABLE FRAGMENTS
break in prefrence to those that form less stable fragments
Types of IONIZATION SOURCES
EI = Electric Ionization
HARD ionization process
CI = Chemical Ionization
soft ionization process
- *MALDI =** Matrix-Assisted laser desorption ionization
- soft ionization w/ assist of matrix*
ESI = Electrospray ionization
coupled to HPLC, Liquid sample passed through HIGH VOLTAGE
DESI = Desorption electrospray ionization
analysis DIRECTLY off surface, quick and fast.
Which type of ionization technique is a
HARD ionization process & what is a hard ionization process?
ELECTRONIC IONIZATION = EI
much EXCESS energy and possibility of
extensive fragmentation
that gives us Fingerprint Information
vaporized compound, we sometimes want this
Which type of ionization technique is a
SOFT ionization process & what is a soft ionization process?
MALDI is also a “soft” ionization
CHEMICAL IONIZATION = CI
less energy,
molecular ion predominates, NOT VERY FRAGMENTED
has to be a volatile compound
provides molecular MASS information / MW
MALDI
type of Ionization Technique
Matrix-Assisted Laser Desorption Ionization
The MATRIX ABSORBS/ASSISTS large amounts of energy
and transfers some of theis energy to the substrate
to MINIMIZE substrate DECOMPOSITION
SOFT IONIZATION
substrate does not decompose much due to the MATRIX
ESI
type of ionization technique
Electrospray Ionization
liquid sample is passed through a HIGH VOLTAGE metal capillary
COUPLED TO HPLC
nebulized & carried by Nitrogen Gas
to produce an aerosol of charged droplets
DESI
type of ionization technique
Desorption Electrospray Ionization
combines real-time microscale solvent etraction in
fast movign droplets with ESI
MS analysis DIRECTLY OFF OF SURFACES
quick & direct!
Types of Mass Analyzers
MS
Magnetic Sector Analyzer
uses a magnetic field, different size = different trajectory
Quadrupole Analyzer
seperated by passing through a MASS FILTER, different size = diff detection
Time-of-flight (TOF) Analyzer
ions are seperated according to their VELOCITIES
What do MASS Analyzers do?
After ions are generated they must be SEPERATED & DETECTED
Based on mass to charge ratio = m/z
ions behave differently in ELECTRIC or MAGNETIC fields
Magnetic Sector Analyzer
type of Mass Analyzer for MS
New ions are passed through a MAGNETIC FIELD
the radius of curvature : m/z
allolows for distinction between HEAVY & LIGHTER IONS
VARY IN TRAJECTORY
Different size = different trajectory
Quadrupole Analyzer
type of Mass Analyzer for MS
Ions are seperated by passing them through a
quadrupole mass filter = 4 poles/parallel rods
that create a ELECTRIC FIELD around the ion’s flight path
Different Size = Different detection
Time-of-Flight Analyzer
TOF
type of Mass Analyzer for MS
Ions are seperaed according to their
VELOCITIES
generated in a short burst –> then accelerated
adv = speed of detection / sensitivity / unlimited mass range
- *Time taken to traverse a SPECIFIC PATH** depends on the
- *MASS of the ion**
typically employed with MALDI
What is USELESS to detect in COSY?
Only shows H-H bonding next to similar Bonds
Does not show what H-C connectivity
What is USELESS for HSQC?
Quaternary Carbons
Resonances due to -OH or -NH
>1 bonds away
Showing H-H connectivity
Stereochemistry / configuration
Only tells us which H is DIRECTLY connected to WHICH C
Heteronuclear Single Quantum Coherence
DIRECT H-C, 1* Bonds
What is USELESS for HMBC?
- *Initial identification,**
- mainly for piecing the fragments together*
H-H bonds or 1* bond correlations
Heteronuclear Multiple Bond Correlation
H-C heteronuclear spin coupling
for 2-3 bond correlations, further away
What is USELESS for NOESY
does not read through bond interactions
only goo for through SPACE interactions
Nuclear Overhauser Enhancement SpectroscopY
H-H for stereochemistry / config of double=bonds / stereoconfiguration
Imaging Mass Spectrometry
IMS
Provides a 2-3 Dimensional visualization of the distribution of chemicals from biological samples
Spatial mapping of molecules over a phenotype
for MOLECULAR IMAGES
allows for
distinction of different molecular species
like B-amyloid Plaques in ALZHEIMERS
